JP2004229642A - Protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein based on a physiological activity by analyzing base sequences of cDNA clones contained in a catalogued full-length cDNA library and specifying the physiological activity of the protein encoded with the cDNA clones for those having new sequence and to provide a method for utilizing the DNA encoding the protein. <P>SOLUTION: The protein is (a) a protein comprising a specific amino acid sequence or (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and/or added in the amino acid sequence and having a DNA-binding activity. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０１６０】
ｄｎａｆｏｒｍ３４８３７は、ＦＡＮＴＯＭ Ｎｏ：９５３００４９Ｃ１５をプローブとした発現解析により、膵臓でのみ発現が増加していることがわかった。ｄｎａｆｏｒｍ３３３８３は、それ自身をプローブとした発現解析により、広い組織範囲で発現が低下していることがわかった。ｄｎａｆｏｒｍ３９５３０は、ｄｎａｆｏｒｍ３３３８３をプローブとした発現解析により、広い組織範囲で発現が低下していることがわかった。ｄｎａｆｏｒｍ３４９１６は、それ自身をプローブとした発現解析により、精巣でのみ発現が低下していることがわかった。
【０１６１】
実施例６ ＰＣＲ法を用いた組織発現解析
本発明のタンパク質をコードするｍＲＮＡの正常マウスおよび疾患マウスでの組織発現変動を検討するために、定法（Ｈｉｇｕｃｈｉ Ｒ， ｅｔ ａｌ．， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， １１： １０２６−３０ （１９９３））に従い、ＰＣＲ法を用いた組織発現解析を行った。
【０１６２】
（１）ｃＤＮＡ合成
以下のマウス（森脇和郎、外１名編、Ｍｏｌｅｃｕｌａｒ Ｍｅｄｉｃｉｎｅ別冊、Ｖｏｌ． ３６「自然発症疾患モデル動物 」、中山書店、１９９９年）の１９組織からトータルＲＮＡを抽出し、オリゴｄＴをプライマーとして逆転写酵素を用いてｃＤＮＡ合成を行った。
（ａ）正常マウスの組織および糖尿病モデルマウスの組織
▲１▼対照マウスＣ５７ＢＬ／ＫｓＪ − ＋ｍ／＋ｍ Ｊｃｌ（メス、８週齢）の全脳、視床、肺、腎臓、骨髄、膵臓、脂肪細胞、肝臓、眼
▲２▼糖尿病モデルマウスＣ５７ＢＬ／ＫｓＪ − ｄｂ／ｄｂ Ｊｃｌ（メス、８週齢）の膵臓、脂肪細胞、肝臓、眼
（ｂ）老化促進マウスの組織
▲１▼正常老化マウス ＳＡＭ Ｒ１／ＴＡ Ｓｌｃ（オス、１３週齢）の海馬、前頭葉皮質
▲２▼老化促進マウス ＳＡＭ Ｐ８／Ｔａ Ｓｌｃ（オス、１５週齢）の海馬、前頭葉皮質
（ｃ） 癌転移モデルマウスの組織
▲１▼対照マウスＢａｌｂ／ｃ（メス、５週齢）の正常結腸
▲２▼癌転移モデルマウスＢａｌｂ／ｃ（メス、６週齢）の結腸癌（マウス腹腔に結腸癌細胞Ｃｏｌｏｎ２６を移植し、２週間後に結腸癌を摘出）
【０１６３】
（２）ＰＣＲ法による定量
下記の６個の、本発明のタンパク質をコードしているｍＲＮＡの発現は、ライトサイクラー定量ＰＣＲ装置（ロシュ・ダイアグノスティクス社）とＬｉｇｈｔＣｙｃｌｅｒ−ＦａｓｔＳｔａｒｔ ＤＮＡマスターＳＹＢＲ Ｇｒｅｅｎ Ｉ試薬を用いて、製品に添付されているプロトコールに従い定量した。定量ＰＣＲに用いた合成ＤＮＡ配列を以下に示す。
【０１６４】
（ａ）ｄｎａｆｏｒｍ３３７７３
５’側プライマー：ＡＧＧＡＴＴＧＧＣＧＡＡＣＴＡＴＣＣＡＧ（配列番号３７）
３’側プライマー：ＣＣＡＣＧＡＧＴＧＡＡＣＡＴＴＴＧＣＡＴ（配列番号３８）
（ｂ）ｄｎａｆｏｒｍ３８８６１
５’側プライマー：ＡＣＴＣＡＧＡＧＡＧＣＣＡＧＣＣＡＧＡＡ（配列番号３９）
３’側プライマー：ＴＧＴＴＧＧＡＡＣＣＧＴＴＴＣＣＴＧＡＧ（配列番号４０）
（ｃ）ｄｎａｆｏｒｍ４１１４３
５’側プライマー：ＧＣＴＧＣＡＣＡＡＡＣＧＧＴＣＴＣＡＴＡ（配列番号４１）
３’側プライマー：ＡＣＣＡＡＧＡＧＧＴＧＣＡＡＣＡＧＡＧＧ（配列番号４２）
（ｄ）ｄｎａｆｏｒｍ４２５１５
５’側プライマー：ＴＧＴＧＣＴＧＴＣＡＴＣＴＧＡＧＡＣＴＴＧＡ（配列番号４３）
３’側プライマー：ＣＣＴＴＧＴＡＣＡＣＡＡＣＣＡＡＧＧＧＴＡＧＡ（配列番号４４）
（ｅ）ｄｎａｆｏｒｍ５１２１８
５’側プライマー：ＡＧＧＡＴＴＧＧＣＧＡＡＣＴＡＴＣＣＡＧ（配列番号４５）
３’側プライマー：ＣＴＣＣＡＣＧＡＧＴＧＡＡＣＡＴＴＴＧＣ（配列番号４６）
（ｆ）ｄｎａｆｏｒｍ６０４４１
５’側プライマー：ＴＣＡＧＧＡＡＡＣＧＧＴＴＣＣＡＡＣＡＴ（配列番号４７）
３’側プライマー：ＴＣＣＴＴＧＧＡＧＧＡＴＴＴＣＴＴＣＴＣＴＧ（配列番号４８）
【０１６５】
定量結果はＧｌｙｃｅｒａｌｄｅｈｙｄｅ ３−ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）を内部標準として、補正した。即ち、各組織での対象遺伝子の発現量（コピー数／μｌ）をＧＡＰＤＨの発現量（コピー数／μｌ）で除し、定数（１×１０^６）（注：１０の６乗）を乗して表示した。
【０１６６】
結果をまとめると、ｄｎａｆｏｒｍ３３７７３は全身で強く発現するが、特に肺、膵臓、脂肪、脳で強力に発現した。ｄｎａｆｏｒｍ３８８６１は全身で強く発現するが、特に膵臓、肺で強力に発現した。ｄｎａｆｏｒｍ４１１４３は全身で発現するが、膵臓、肺、脂肪で比較的強く発現した。ｄｎａｆｏｒｍ４２５１５は脳に特異的に発現し、特に前頭様皮質で強力に発現した。ｄｎａｆｏｒｍ５１２１８は全身で強く発現するが、特に肺、膵臓、脂肪、脳で強力に発現した。ｄｎａｆｏｒｍ６０４４１は全身で発現するが、特に膵臓、肺で強く発現した。上記クローンのｃＤＮＡおよび該ｃＤＮＡによってコードされるタンパク質は、糖尿病や癌などの治療や診断に応用できる。また該ｃＤＮＡによってコードされるタンパク質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性がある。
【０１６７】
【表２】

【０１６８】
実施例７ タンパク質−タンパク質相互作用解析
哺乳動物細胞におけるｔｗｏ−ｈｙｂｒｉｄ法（Ｓｕｚｕｋｉ， Ｈ．， ｅｔ ａｌ．， Ｇｅｎｏｍｅ Ｒｅｓｅａｒｃｈ， １１， １７５８−１７６５ （２００１））を用いて、２種類のマウス全長ｃＤＮＡの塩基配列（ｄｎａｆｏｒｍ３３７７３、ｄｎａｆｏｒｍ３４８３７）のタンパク質コード配列がコードするタンパク質のタンパク質−タンパク質相互作用を網羅的に解析した。
（１）ＰＣＲ法を用いた迅速なサンプル調製
哺乳動物細胞でのｔｗｏ−ｈｙｂｒｉｄ実験は、ＣｈｅｃｋＭａｔｅ ｍａｍｍａｌｉａｎ ｔｗｏ−ｈｙｂｒｉｄ ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社製）を利用した。タンパク質−タンパク質相互解析用のサンプルは、ＣＭＶプロモーターの下流にＧａｌ４遺伝子のＤＮＡ結合領域を挿入したプラスミドベクターｐＢＩＮＤ、ＣＭＶプロモーターの下流にＶＰ１６遺伝子の転写活性化領域を挿入したプラスミドベクターｐＡＣＴ，および５個のＧａｌ４結合領域とＴＡＴＡボックスの下流にレポーターであるルシフェラーゼ遺伝子を挿入したプラスミドベクターｐＧ５ｌｕｃを鋳型として調製した。Ｇａｌ４遺伝子と２種類のマウス全長ｃＤＮＡの塩基配列（ｄｎａｆｏｒｍ３３７７３、ｄｎａｆｏｒｍ３４８３７）のタンパク質コード配列との融合遺伝子、並びにＶＰ１６遺伝子とマウスｃＤＮＡライブラリーＦＡＮＴＯＭ（ｈｔｔｐ：／／ｆａｎｔｏｍ．ｇｓｃ．ｒｉｋｅｎ．ｇｏ．ｊｐ／）の各クローンが有する完全長ｃＤＮＡのタンパク質コード配列との融合遺伝子は、基本的にＰｒｏｍｅｇａ社のプロトコールに従い共通配列部分を用いた連結と２段階ＰＣＲ法を組み合わせて作成した（Ｓｕｚｕｋｉ， Ｈ．， ｅｔ ａｌ．， Ｇｅｎｏｍｅ Ｒｅｓｅａｒｃｈ， １１， １７５８−１７６５ （２００１） ）。マウスｃＤＮＡのタンパク質コード配列を、５’側に共通配列をもち３’側に遺伝子特異的な配列をもつフォワードプライマーおよびＭ１３ユニバーサルプライマーとを用いてＰＣＲ増幅した後、上記増幅産物とｐＢＩＮＤまたはｐＡＣＴのＰＣＲ増幅産物（３’側に共通配列を付加した）とを混和し、それぞれネスティドプライマーを用いて第２段のＰＣＲ増幅を行い、Ｇａｌ４とマウスタンパク質の融合タンパク質を発現させるベクター（ＢＩＮＤサンプル）またはＶＰ１６とマウスタンパク質の融合タンパク質を発現させるベクター（ＡＣＴサンプル）を構築した。
【０１６９】
（２）ハイスループットな哺乳動物細胞でのｔｗｏ−ｈｙｂｒｉｄ実験
ＰＣＲ法で調製したＢＩＮＤおよびＡＣＴサンプルは、それ以上の精製を行わずに直接使用した。ＢＩＮＤサンプルおよびＡＣＴサンプルのそれぞれ０．２５μｌ、３０ｎｇのｐＧ５ｌｕｃ、および９．５μｌのＯｐｔｉ−ＭＥＭ培地（Ｌｉｆｅｔｅｃｈ社製）を３８４ウェルプレートに分注した。Ｏｐｔｉ−ＭＥＭ培地にて３２倍希釈したＬＦ２０００トランスフェクション試薬（Ｌｉｆｅｔｅｃｈ社製）１０μｌをウェルに加えて混和し２０分間インキュベーション後、Ｆ１２培地にて１，３００細胞／μｌに懸濁したＣＨＯ−Ｋ１チャイニーズハムスター細胞液２０μｌを加えて良く懸濁した。アッセイサンプルをＣＯ_２インキュベーター内で２０時間培養後、ルシフェラーゼ活性はＳｔｅａｄｙ−Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社製）を用いて測定し、相互作用を確認した。
【０１７０】
この結果、２種類のマウス全長ｃＤＮＡの塩基配列（ｄｎａｆｏｒｍ３３７７３、ｄｎａｆｏｒｍ３４８３７）のタンパク質コード配列がコードするタンパク質は、以下に示す特定のタンパク質（マウスｃＤＮＡライブラリーＦＡＮＴＯＭの特定のクローンが有するｃＤＮＡのタンパク質コード配列がコードする特定のタンパク質）との相互作用をそれぞれ有していることが明らかとなった。
【０１７１】
【表３】

【０１７２】
（３）考察
Ｄｎａｆｏｒｍ３３７７３にコードされる本発明のタンパク質は、ＳＲＰ４０と分子間相互作用することがわかった。ＳＲＰ４０は、ｍＲＮＡ前駆体のスプライシング反応に関与する蛋白質の一群であるＳＲ蛋白質のひとつであって、ｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ ３−ｋｉｎａｓｅが関与するｐｒｏｔｅｉｎ ｋｉｎａｓｅ Ｃ ｂｅｔａ ＩＩ ｍＲＮＡのスプライシング経路に必要な蛋白質である。このＳＲＰ４０がｐｒｏｔｅｉｎ ｋｉｎａｓｅ Ｃ ｂｅｔａ ＩＩの発現を調節し、この働きによりｉｎｓｕｌｉｎ依存的な糖代謝が制御されていることが知られている（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．， ２７６（２５）：２２６４８−５４， ２００１） 。したがって、ＳＲＰ４０の異常は糖尿病の原因となりうる。Ｄｎａｆｏｒｍ３３７７３にコードされる本発明のタンパク質は、Ｚｉｎｃｆｉｎｇｅｒをもち、転写制御活性を有することが予想されており、これらの結果を総合的に解析すると、糖尿病の原因となりうるＳＲＰ４０と結合するＤｎａｆｏｒｍ３３７７３にコードされるタンパク質は、糖尿病の原因となりうる蛋白質であると推測できる。
Ｄｎａｆｏｒｍ３４８３７にコードされる本発明のタンパク質は、Ｐｒｅｇｎａｎｅ Ｘ ｒｅｃｅｐｔｏｒ ２（ＰＸＲ２）と相互作用することがわかった。ＰＸＲ２は核内ステロイド受容体の１つで、ステロイド化合物を介する情報伝達系に複合的に関与する核内受容体の１つである。肝臓においては細胞質に存在し、リガンドに曝露されることによりＲＸＲ（Ｒｅｔｉｎｏｉｄ Ｘ ｒｅｃｅｐｔｏｒ）とヘテロ二量体を形成して核に移行し、特異的遺伝子発現に関与していることで知られている。具体的には、例えば、ＰＸＲは医薬品に応答してチトクロームＰ４５０（ＣＹＰ）酵素等の薬物代謝系を誘導することが知られており、生体内での異物（医薬品）センサーとして機能していると推測されている。また、このＰＸＲの異常は、糖尿病、高脂血症、高血圧、虚血性心疾患といったいわゆる生活習慣病の原因となり、様々な内分泌疾患を引き起こすことが知られている。Ｄｎａｆｏｒｍ３４８３７にコードされる本発明のタンパク質はＺｉｎｃ−ｆｉｎｇｅｒ ｐｒｏｔｅｉｎであり、転写制御活性を有する蛋白質であることが予測されており、核内に存在して特定のＤＮＡ配列に結合することによって情報伝達系に関与していることが予想される。以上の結果を総合的に解析すると、Ｄｎａｆｏｒｍ３４８３７は、ＰＸＲ２と共存する条件でヘテロ二量体を形成し、転写制御に関与する可能性があるタンパク質であると推測できる。
【０１７３】
実施例８ ヒトオルソログＤＮＡの取得
（１）ｄｎａｆｏｒｍ３３３８３のヒトオルソログＤＮＡの予測
ｄｎａｆｏｒｍ３３３８３の塩基配列（配列番号３）を問い合わせとして、ヒトゲノムドラフト配列（ＮＣＢＩ Ｂｕｉｌｄ ３０；ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ａｂｏｕｔ／Ｄｏｃ／ｈｓ＃ｇｅｎｏｍｅｉｎｔｒｏ．ｈｔｍｌ）に対してＢＬＡＳＴ検索を行ったところ、相同性の高い領域として１６番染色体の６ｐ１２．１の２４．６２Ｍｂから２４．６６Ｍｂの領域を見出した。
該ゲノム配列領域に対して、遺伝子予測プログラムＧｅｎｓｃａｎ（ｈｔｔｐ：／／ｇｅｎｅｓ．ｍｉｔ．ｅｄｕ／ＧＥＮＳＣＡＮ．ｈｔｍｌ） を用いて遺伝子領域予測を行い、得られた予測転写産物配列に対して相同性検索を行ったところ、３５２０塩基よりなる配列（配列番号４９）を得た。この塩基配列は、塩基番号３１９−３２１９に２９０４塩基のオープンリーディングフレーム（配列番号３５）を含んでいた。配列番号３のマウス塩基配列は、配列番号３５のヒト塩基配列と約２５８０塩基対の長さに渡って８１％の相同性をもつことがわかった。
【０１７４】
また、配列番号３５の塩基配列から配列番号３６のヒトアミノ酸配列に翻訳されると予測された。配列番号３６のアミノ酸配列とｄｎａｆｏｒｍ３３３８３のオープンリーディングフレームから予測されるアミノ酸配列（配列番号１６）との間には、８６４アミノ酸配列に渡って８１％の相同性があった。
さらに、配列番号３６のアミノ酸配列を問い合わせとして、マウスｃＤＮＡライブラリー ＦＡＮＴＯＭデータベース（ｈｔｔｐ：／／ｆａｎｔｏｍ．ｇｓｃ．ｒｉｋｅｎ．ｇｏ．ｊｐ／）に対してＢＬＡＳＴによる相同性検索を行ったところ、配列番号１６のアミノ酸配列が最も相同性が高かった。
【０１７５】
なお、公開塩基配列データベースであるｅｍｂｌｅデータベースと特許データベースであるＧｅｎｓｅｑデータベースに対してのＢＬＡＳＴ相同性検索においては、配列番号３５の塩基配列よりも相同性の高いヒト塩基配列は検索できなかった。
従って、配列番号３５が、配列番号３に対する新規なヒトオルソログＤＮＡであると考えられた。
（２）取得されたヒトオルソログＤＮＡの塩基配列の解析
上記（１）で予測されたヒトオルソログＤＮＡの塩基配列および該塩基配列によりコードされるアミノ酸配列について、さらなる解析を行った。
まず、配列番号３６に示されるアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）および特許配列のデータベース中には、配列番号１６に記載のマウスのアミノ酸配列以上に相同性を示す配列はなかった。このことからも、配列番号３６に記載のアミノ酸配列を有するタンパク質は、配列番号１６に記載のアミノ酸配列配列を有するマウスタンパク質のヒトオルソログタンパク質であることが推測された。
配列番号３６に記載のヒトアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号３９−１３４にＳＣＡＮ ｄｏｍａｉｎ（ＰｆａｍにＳＣＡＮとしてエントリーされるアミノ酸配列）を、アミノ酸番号２２９−２６９にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎ（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）を、アミノ酸番号７７５−７９７、８０３−８２５、８３１−８５３、８５９−８８１、８８７−９０９、９１５−９３７にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。
ＳＣＡＮ ｄｏｍａｉｎは一部のＣ２Ｈ２タイプのＺｎフィンガータンパク質のＮ末端に見られる保存されたモチーフで、オリゴマー形成の調節と転写制御の役割をもつことが知られている。またＫｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）はＣ２Ｈ２タイプのＺｎフィンガータンパク質の約１／３に見られ、Ｎ末端部分に存在し、ＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
以上のことから、配列番号３６に記載のアミノ酸配列を有するタンパク質は、Ｋｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）を持つＣ２Ｈ２タイプのＺｎフィンガータンパク質で、転写の抑制にかかわる転写因子であることが推測された。
【０１７６】
（３）ｄｎａｆｏｒｍ３３３８３のヒトオルソログＤＮＡの取得
上記（１）および（２）において新規なヒトオルソログＤＮＡが予測されたことから、以下のとおり、このｃＤＮＡのクローニングをおこなった。
まず、このｃＤＮＡを取得する遺伝資源となる組織を選択するために、フォワードプライマー１（配列番号５０）とリバースプライマー１（配列番号５１）を用いてヒト組織由来ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ社製）を鋳型としたＰＣＲを行ったところ、子宮、前立腺、脳下垂体、脳、胎児脳、海馬、視床で目的とする約１５０ｂｐのＤＮＡの増幅が確認された。フォワードプライマー１は配列番号３５の塩基番号１２９４−１３１３に相当し、リバースプライマー１は配列番号３５の塩基番号１４４３−１４２４に相当する。
【０１７７】
次に、オープンリーディングフレーム全長を取得するために、これら組織由来のｃＤＮＡを鋳型としてフォワードプライマー２（配列番号５２）とリバースプライマー２（配列番号５３）を用いたＰＣＲを行った。これらのプライマーは、配列番号４９のヒト塩基配列においてオープンリーディングフレームの外側に位置する配列を用いて設計されたプライマーであって、フォワードプライマー２は配列番号４９の塩基番号２５３−２７２に相当し、リバースプライマー２は配列番号４９の塩基番号３２５６−３２３７に相当する。
【０１７８】
ＰＣＲの結果、子宮、前立腺、脳下垂体、胎児脳、海馬、視床で予測値と一致する２．９ｋｂのＤＮＡが増幅した。これらのうち子宮、前立腺、胎児脳に由来するＰＣＲ増幅ＤＮＡ断片を鋳型に直接シークエンスを行ったところ、３種とも同じ配列を有しており、ＰＣＲによるミスはないことがわかった。そこで、子宮由来のＰＣＲ増幅ＤＮＡ断片をｐＣＲ４Ｂｌｕｎｔｉｎｇ−ＴＯＰＯベクター（Ｉｎｖｉｔｒｏｇｅｎ社製）にライゲーションして、得られた独立のコロニー３個から由来するプラスミドのシークエンスをおこなったところ、３つとも配列番号４９の塩基番号２５３−３２５６（配列番号３５に記載された、ヒトのオープンリーディングフレーム全長に相当する塩基配列を含む）と全く同じ配列が挿入されていた。
以上のことから、予想されたヒト転写物（タンパク質）が実際に発現していることが示され、そのｃＤＮＡを取得することができたことがわかった。かくして取得されたヒトホモログＤＮＡを、ＺＧ１００１と名付けた。
【０１７９】
このヒト塩基配列に由来するアミノ酸配列（配列番号３６）とマウス塩基配列に由来するアミノ酸配列（配列番号１６）とを比較すると、ヒトアミノ酸配列上のアミノ酸番号６４−９１３と、マウスアミノ酸配列上のアミノ酸番号７−８５６の部分で一致度が高く、Ｎ末端はヒトアミノ酸配列の方が５９アミノ酸長く、Ｃ末端は３６アミノ酸長かった。
さらに、ヒト塩基配列（配列番号３５）とマウス塩基配列（配列番号３）をそれぞれヒトゲノム、マウスゲノムにマッピングすると、両者とも７つのエクソンにコードされていた。それぞれのＮ末端、Ｃ末端をコードする部分は、一致度が高い部分から一致度が低い部分にかけてひとつのエクソンであった。すなわち、これらの配列のＮ末端及びＣ末端の違いは、エクソンの使い分けではなく種による違いであることがわかった。
【０１８０】
実施例９ 標的転写調節領域に対する結合能の解析
（１）無細胞蛋白質合成系を用いた本発明の蛋白質の調製
本発明のタンパク質をコードするＤＮＡを含むプラスミドに対して、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９９，１４６５２−１４６５７ （２００２）に記載されている方法に準じてＰＣＲ反応を行い、転写用のＤＮＡ断片を調製した。このＤＮＡを鋳型としてＳＰ６ ＲＮＡ Ｐｏｌｙｍｅｒａｓｅ（Ｐｒｏｍｅｇａ社製）を用いて転写反応を行ない、ｍＲＮＡを合成し、エタノール沈殿操作により得られたｍＲＮＡを精製した。このｍＲＮＡを用いたタンパク合成は、特開２００２−２０４６８９号公報、およびＰｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９９：１４６５２−１４６５７（２００２）に準じた重層法による無細胞蛋白質合成系を用いて行った。重層法無細胞蛋白質合成系にて使用する翻訳溶液（２５μｌ）には、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９７：５５９−５６４（２０００）に従って調製された６μｌの小麦胚芽抽出液および上述したｍＲＮＡ（０．０２ｎｍｏｌ）を添加して用い、その組成は２４ ｍＭ Ｈｅｐｅｓ／ＫＯＨ（ｐＨ ７．８）， １．２ ｍＭ ＡＴＰ， ０．２５ ｍＭ ＧＴＰ， １６ ｍＭ ｃｒｅａｔｉｎｅ ｐｈｏｓｐｈａｔｅ， １０ μｇ ｃｒｅａｔｉｎｅ ｋｉｎａｓｅ， ｒｉｂｏｎｕｃｌｅａｓｅ ｉｎｈｉｂｉｔｏｒ（２０ｕｎｉｔｓ）， ２ ｍＭ ＤＴＴ， ０．４ ｍＭ ｓｐｅｒｍｉｄｉｎｅ， ０．３ ｍＭ Ｌ型アミノ酸（２０種）， ２．７ ｍＭ ｍａｇｎｅｓｉｕｍ ａｃｅｔａｔｅ， １００ ｍＭ ｐｏｔａｓｓｉｕｍ ａｃｅｔａｔｅ， ５ μｇ 小麦胚芽由来ｔＲＮＡ， ０．０５％ Ｎｏｎｉｄｅｔ Ｐ−４０および０．００５％ ＮａＮ３から成る。また翻訳用緩衝液は３１．３ｍＭ ＨＥＰＥＳ／ＫＯＨ（ｐＨ７．８） ， ２．６７ｍＭ Ｍｇ（ＯＡｃ）２ ， ９３ｍＭ ＫＯＡｃ ， １．２ｍＭ ＡＴＰ， ０．２５７ｍＭ ＧＴＰ ， １６ｍＭ ｃｒｅａｔｉｎｅ ｐｈｏｓｐｈａｔｅ， ２．１ｍＭ ＤＴＴ， ０．４１ｍＭ ｓｐｅｒｍｉｄｉｎｅ， ０．３ｍＭＬ型アミノ酸（２０種）， １μＭ Ｅ−６４ ， ０．００５％ ＮａＮ３ ， ０．０５％ ＮＰ−４０から成る。重層法による無細胞蛋白質合成は、まず９６穴プレートに翻訳用緩衝液を１２５μｌずつ加えて、この翻訳用緩衝液が入ったそれぞれの穴に底からゆっくりと翻訳溶液を重層し、このプレートを２６℃インキュベーターで保温して１６時間反応させることにより行った。
【０１８１】
（２）標的配列を固定化したセンサーチップを用いたＳＰＲ測定方法
ＢＩＡａｐｐｌｉｃａｔｉｏｎｓ ｈａｎｄｂｏｏｋ， ｃｈａｐｔｅｒ４．４の記載に従い、センサーチップ表面にビオチン化した以下の５３種類の二重鎖ＤＮＡを別々に固定化した。センサーチップはＳＡタイプ（ビアコア社製）を用い、ＳＰＲ測定および解析は、ＢＩＡＣＯＲＥ３０００（ビアコア社製）を用いた。
【０１８２】
公知の転写因子と、設計した標的転写調節領域のＤＮＡまたはＤＮＡ断片の塩基配列（以下、これを「標的配列」と称することがある）、または、複数の転写因子が共通して認識する標的転写調節領域のＤＮＡまたはＤＮＡ断片の塩基配列（以下、これを「コンセンサス標的配列」と称することがある）の関係は、次の通りである。
［１］ｖ−ｊｕｎ， ｃ−ｊｕｎ， ｊｕｎＢ， ｊｕｎＤ， ｄＪＲＡ， ｃ−ｆｏｓ， ｆｏｓＢ１， ｆｏｓＢ２， Ｆｒａ−１， ＬＲＦ−１， ｖ−ｍａｆ， ｍａｆＧ， ＮＦ−Ｅ２ ｐ４５， ａＮＦ−Ｅ２， ｆＮＦ−Ｅ２， Ｎｒｆ ｓｈｏｒｔ ｆｏｒｍ， ＧＣＮ４， ｙＡＰ−１， ＣＲＥＢ−２， ＡＴＦ−３， ＣＲＥ−ＢＰ１， ＣＲＥ−ＢＰ３， ＡＴＦ−ａ， ＣＲＥＢ−３４１， ＣＲＥＢ−３２７， ＣＲＥＭ， ｄＣＲＥＢ２， ｄＣＲＥＢ２−ｂ， ｄＣＲＥＢ２−ｃ， ｄＣＲＥＢ２−ｄ， ｄＣＲＥＢ２−ｑ， ｄＣＲＥＢ２−ｒ， ｄＣＲＥＢ２−ｓのコンセンサス標的配列：ＴＧＡＴＧＡＣＧＴ（配列番号５４）
［２］Ｃ／ＥＢＰａｌｐｈａ， Ｃ／ＥＢＰｂｅｔａ， ｐ３４Ｃ／ＥＢＰｂｅｔａ， ＣＨＯＰ−１０のコンセンサス標的配列：ＡＡＧＴＧＧＣＧＡＡＡＧＡＧＡＣＡ（配列番号５５）
［３］ＶＢＰ， Ｈｌｆ， ＣＰＲＦ−２， ＥｍＢＰ−１ｂ， ＥｍＢＰ−１ｂ， ＧＢＦ１， ＧＢＦ２， ＧＢＦ３， ＣＰＲＦ−１， ＴＡＦ−１， ＨＢＰ−１ａ， ＧＢＦ９， ＧＢＦ１， ＧＢＦ１２， ＣＰＲＦ−３， ＴＧＡ１ａ， ＴＧＡ１ｂ， Ｏ２， ＳＴＥ４のコンセンサス標的配列：ＡＧＡＡＧＣＡＣＧＴＧＧ（配列番号５６）
［４］ＯＰＩ１， Ｅ２Ａ， Ｅ４７， ＩＴＦ−２／ＳＥＦ２−１Ｂ， ＳＥＦ−１Ａ， ＭｙｏＤ， ｐ４２Ｔａｌ−１のコンセンサス標的配列：ＡＡＣＡＧＡＴＧＧＴ（配列番号５７）
［５］ＨＥＮ−１の標的配列：ＧＧＧＧＣＧＣＡＧＣＴＧＣＧＧＣＣＣ（配列番号５８）
［６］ＡｈＲ， Ａｒｎｔ のコンセンサス標的配列：ＧＧＧＧＡＴＴＧＣＧＴＧ（配列番号５９）
［７］ＵＳＦの標的配列：ＧＴＣＡＣＧＴＧＧＴ（配列番号６０）
［８］ＮＦ−１Ａ１， ＮＦ−１Ａ１．１， ＮＦ−１Ａ６， ＮＦ−１Ｂ１， ＮＦ−１Ｂ１， ＮＦ−１Ｂ２， ＮＦ−１Ｃ２／ＣＴＦ−２， ＣＴＦ−４， ＣＴＦ−６のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号６１）
［９］ＲＦ−Ｘ１の標的配列：ＧＧＴＡＡＣＡＴＡＧＣＡＡＣ（配列番号６２）
［１０］ＡＰ２ａｌｐｈａＡ／ＡＰ−２ａｌｐｈａ１， ＡＰ２ａｌｐｈａ２， ＡＰ２ａｌｐｈａ３， ＡＰ２ａｌｐｈａ４， ＡＰ２ａｌｐｈａＢ， ＡＰ２ｂｅｔａ， ＡＰ２ｇａｍｍａのコンセンサス標的配列：ＣＧＣＣＣＣＣＣＧＧＣＧ（配列番号６３）
［１１］ＧＲの標的配列：ＧＧＴＡＣＡＡＡＡＴＧＴＴＣＴ（配列番号６４）
［１２］ＡＲの標的配列：ＡＡＣＡＴＴＡＴＧＴＴＣＴ（配列番号６５）
［１３］ＥＲの標的配列：ＡＡＧＧＧＡＡＡＡＴＧＡＣＣＣＣＣ（配列番号６６）
［１４］ＲＸＲ−ａｌｐｈａの標的配列：ＧＧＴＣＡＴＡＧＧＧＧＴ（配列番号６７）
［１５］ＰＰＡＲａｌｐｈａの標的配列：ＣＴＡＧＧＧＣＡＡＡＧＧＴＣＡ（配列番号６８）
［１６］ＰＰＡＲｇａｍｍａの標的配列：ＧＧＴＣＡＡＡＧＧＴＣＡ（配列番号６９）
［１７］ＣＯＵＰ−ＴＦ１，ＨＮＦ−４ａｌｐｈａ１， ＨＮＦ−４ａｌｐｈａ２の標的配列：ＴＧＡＡＣＴＴＴＧＡ（配列番号７０）
［１８］ＣＦ１の標的配列：ＧＧＧＧＴＣＡＣＣ（配列番号７１）
［１９］ＧＡＴＡ−１， ＧＡＴＡ−２， ＧＡＴＡ−３， ＧＡＴＡ−４のコンセンサス標的配列：ＣＣＡＧＡＴＡＡＧＧ（配列番号７２）
［２０］ＡＲＥＡ／ＮＩＴ−２の標的配列：ＴＡＴＣＴＣ（配列番号７３）
［２１］Ｓｐ１の標的配列：ＧＧＧＧＧＧＧＧＧＧ（配列番号７４）
［２２］ＹＹ１の標的配列：ＣＧＧＣＣＡＴＣＴＴＧＧＣＴ（配列番号７５）
［２３］Ｅｇｒ−１， Ｅｇｒ−２， Ｅｇｒ−３のコンセンサス標的配列：ＴＧＣＧＴＧＧＧＣＧ（配列番号７６）
［２４］Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号７７）
［２５］ＣＦ２−ＩＩの標的配列：ＧＴＡＴＡＴＡＴＡ（配列番号７８）
［２６］Ｅｖｉ−１の標的配列：ＡＧＡＴＡＡＧＡＴＡＡ（配列番号７９）
［２７］Ｉｋａｒｏｓ， ＭＺＦ−１のコンセンサス標的配列：ＴＴＧＧＧＡＧＧ（配列番号８０）
［２８］Ｔｒａｍｔｒａｃｋ６９Ｋの標的配列：ＧＧＡＣＣＴＧＣ（配列番号８１）
［２９］ＨＯＸ９の標的配列：ＴＧＡＣＡＧＴＴＴＡＡＣＧＡ（配列番号８２）
［３０］ＣＤＰの標的配列：ＣＣＡＡＴＡＡＴＣＧＡＴ（配列番号８３）
［３１］ＨＮＦ−１Ａの標的配列：ＧＧＴＴＡＡＴＧＡＴＴＡＡＣＣＡＣ（配列番号８４）
［３２］Ｎｋｘ−２．２， Ｎｋｘ−２．５， ＴＴＦ−１のコンセンサス標的配列：ＴＴＡＡＧＴＧＧＴＴ（配列番号８５）
［３３］Ｏｃｔ−１Ａ， Ｏｃｔ−１Ｂ， Ｏｃｔ−１Ｃ のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号８６）
［３４］Ｏｃｔ−２， Ｏｃｔ−２．１／Ｏｃｔ−２Ｂのコンセンサス標的配列：ＴＡＴＴＴＧＣＡＴ（配列番号８７）
［３５］Ｐａｘ−３， Ｐａｘ−６のコンセンサス標的配列：ＣＧＴＣＡＣＧＣＴＴＧＡ（配列番号８８）
［３６］Ｐａｘ−１の標的配列：ＣＣＧＴＴＣＣＧＣＴＣＴＡＧＡＴＡＴ（配列番号８９）
［３７］ＨＳＦ１（ｓｈｏｒｔ）， ＨＳＦ２， ｄＨＳＦ， ｆｕｎｇａｌＨＳＦのコンセンサス標的配列：ＡＧＡＡＡＡＧＡＡＡＡＧＡＡＡ（配列番号９０）
［３８］ｃ−Ｍｙｂ， Ａ−Ｍｙｂ， ｖ−Ｍｙｂ， Ｐ（ｌｏｎｇ）， Ｐ（ｓｈｏｒｔ）， Ｃ１（ｌｏｎｇ）， Ｃ１（ｓｈｏｒｔ）のコンセンサス標的配列：ＡＡＣＧＧＧＣＣＣ（配列番号９１）
［３９］ｃ−Ｅｔｓ−１＃ｐ５４， Ｅｔｓ−１＃ｄｅｌｔａｉＶ／ＶＩＩ， Ｅｔｓ−２， Ｅｌｋ−１， ＳＡＰ−１， ＳＡＰ−１ｂ， Ｅｒｇ−１， ｐ５５ｅｒｇ， Ｆｌｉ−１ｂ， Ｅ４ＴＦ１−６０／ＧＡＢＰ−ａｌｐｈａ， Ｅ７４Ａのコンセンサス標的配列：ＧＡＣＡＧＧＡＡＧＴＧ（配列番号９２）
［４０］ＩＲＦ−１， ＩＲＦ−２の標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号９３）
［４１］ｐ５０の標的配列：ＧＧＧＧＡＣＴＴＴＣＣ（配列番号９４）
［４２］ＮＦ−ＡＴｃ， ＮＦ−Ａｔｐのコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号９５）
［４３］ｐ９１， ｐ８４のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号９６）
［４４］ＳＴＡＴ２， ＳＴＡＴ３， ＳＴＡＴ４， ＳＴＡＴ５Ａ， ＳＴＡＴ５Ｂ， ＳＴＡＴ６のコンセンサス標的配列：ＴＴＴＣＣＣＧＧＧＡＡＡＴＧ（配列番号９７）
［４５］ｐ５３の標的配列：ＧＧＡＣＡＴＧＣＣＣＧＧＧＣＡＴＧＴＣ（配列番号９８）
［４６］ＭＥＦ−２Ａの標的配列：ＣＴＣＴＡＡＡＡＡＴＡ（配列番号９９）
［４７］ＳＲＦの標的配列：ＣＣＡＴＡＴＡＴＧＧＡＣＡＴ（配列番号１００）
［４８］Ｅ２の標的配列：ＡＡＣＣＡＡＡＡＡＣＧＧＴＡＡ（配列番号１０１）
［４９］ＴＢＰの標的配列：ＴＡＴＡＡＡＡ（配列番号１０２）
［５０］ＳＲＹ， Ｓｏｘ−５， Ｓｏｘ−９の標的配列：ＡＡＡＡＡＡＣＡＡＴＡＧＧＧ（配列番号１０３）
［５１］ｍａｔ−Ｍｃの標的配列：ＴＣＡＴＴＧＴＴ（配列番号１０４）
［５２］ＣＰ１Ａ， ＣＰ１Ｂ， ＣＢＦ−Ｃのコンセンサス標的配列：ＣＴＧＡＴＴＧＧＣＴＡＣＣ（配列番号１０５）
［５３］ＡＭＬ１ａの標的配列：ＴＧＴＧＧＴ（配列番号１０６）
【０１８３】
まず、上記５３種類の標的配列（配列番号５４〜配列番号１０６）を有するＤＮＡに関し、それぞれの５’側にビオチンが付加したＤＮＡとその相補な塩基配列を有するＤＮＡを定法により個別にアニーリングさせ、合計５３種類の二本鎖化したＤＮＡを調製した。一方、解析に用いるセンサーチップは、１枚に付きフローセルが４分割されている。フローセル１は何も固定化せずコントロール区として用い、フローセル２、３および４はそれぞれ上記で調製した二本鎖ＤＮＡを３種類ずつ固定化した。センサーチップのＤＮＡの固定化密度を一定にするため、ＤＮＡ固定化によるＳＰＲ応答値の上昇（ΔＲＵ−ＤＮＡ）をＤＮＡ分子量（ＭＷ）で割った値（Ｄ）が各フローセルで一定になるようにΔＲＵ−ＤＮＡを調節した。同様の要領で、残りのＤＮＡについても固定化を行った。
【０１８４】
以上のようにセンサーチップの作製ができた後、クローンのｃＤＮＡがコードする本発明のタンパク質との結合活性解析を行った。ＳＰＲ法によるＤＮＡとタンパク質間の結合活性解析は既に多数報告がある。本実施例では、Ｍｏｌｅｃｕｌａｒ Ｍｉｃｒｏｂｉｏｌｏｇｙ， ３６（３）， ５５７−５６９（２０００）の測定条件を参考にして行った。まず、フローセル１−２−３−４が直列につながった流路に設定しておく。そこにランニングバッファーを一定流量（５μＬ／ｍｉｎ）で流しておき、ＳＰＲ測定値を安定させた後、各フローセルのベースライン値（ＳＰＲ−ｂａｓｅｌｉｎｅ）を測定する。次に、蛋白質溶液を同流量で流し、蛋白質分子とＤＮＡ鎖との間で特異的結合を形成させる。一定時間注入後、各フローセルのＳＰＲ応答値（ＳＰＲ−ｂｏｕｎｄ）を測定した。
【０１８５】
（４）ＳＰＲ法により得られた測定結果の解析方法
ＳＰＲ応答値からベースライン値を差し引き（［ＳＰＲ−ｂｏｕｎｄ］−［ＳＰＲ−ｂａｓｅｌｉｎｅ］）することにより、真の結合量（Ｂ）を求め、さらに、標準化した値（ｎＢ）を求めた。
測定結果より、ｄｎａｆｏｒｍ３３３８３，３３７３３，３４１９６，３４８３７，３７４７９，３８８６１，３９５３０，４１１４３，４２５１５，５１２１８，５９６３５，６０４４１，６３１６６の１３クローンのｃＤＮＡによってコードされる本発明のタンパク質は、前記［１］〜［５３］の計５３種類の標的配列（配列番号５４〜配列番号１０６）を有するＤＮＡの少なくとも一つに対して結合活性を有していることがわかった。
【０１８６】
実施例１０ ヒト組織発現解析
上記実施例８において、ｄｎａｆｏｒｍ３３３８３についてはヒトオルソログ（ＺＧ１００１）が取得された。そこで、本発明のヒトタンパク質をコードするｍＲＮＡの正常ヒトおよび疾患患者での組織発現変動を検討するために、定法（Ｈｉｇｕｃｈｉ Ｒ， ｅｔ ａｌ．， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， １１： １０２６−３０ （１９９３））に従い、ＰＣＲ法を用いた組織発現解析を行った。
【０１８７】
（１）ｃＤＮＡ合成
以下のヒトの９組織のｍＲＮＡはＣｌｏｎｔｅｃｈ社から購入し、オリゴｄＴをプライマーとした逆転写反応を行いｃＤＮＡを合成した。
正常脳、正常海馬、正常視床、正常腎臓、正常肝臓、正常膵臓、正常骨格筋、正常脂肪、正常脾臓
以下のヒトの８組織のｃＤＮＡはＣｌｏｎｔｅｃｈ社から購入した。
正常乳房、乳癌、正常結腸、結腸癌、正常肺、肺癌、正常胃、胃癌
以下のヒトの２組織のｃＤＮＡはＢｉｏｃｈａｉｎ社から購入した。
正常前頭葉、アルツハイマー病前頭葉
以下のヒトの１組織の全ＲＮＡはユニーテック社から購入し、ｍＲＮＡを抽出後、ｃＤＮＡ合成を行った。
正常末梢血白血球
【０１８８】
（２）ＰＣＲ法による定量
本発明のヒトタンパク質をコードしているｍＲＮＡ（ｄｎａｆｏｒｍ３３３８３のヒトオルソログ ＺＧ１００１のｍＲＮＡ）の発現は、ライトサイクラー定量ＰＣＲ装置（ロシュ・ダイアグノスティクス社製）とＬｉｇｈｔＣｙｃｌｅｒ−ＦａｓｔＳｔａｒｔ ＤＮＡマスターＳＹＢＲ Ｇｒｅｅｎ Ｉ試薬を用いて、製品に添付されているプロトコールに従い定量した。定量ＰＣＲに用いた合成ＤＮＡ配列を以下に示す。
５’側プライマー：ＧＣＴＣＣＧＴＣＣＡＣＴＧＡＴＡＡＡＣＣ（配列番号１０７）
３’側プライマー：ＧＣＧＧＡＴＡＡＡＴＴＣＧＡＴＧＣＣＴＡ（配列番号１０８）
定量結果は、Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ ３−ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）を内部標準として、補正した。即ち、各組織での対象遺伝子の発現量（コピー数／μｌ）をＧＡＰＤＨの発現量（コピー数／μｌ）で除し、定数（１×１０^５）（注：１０の５乗）を乗して表示した。その結果を表４に示す。
【０１８９】
【表４】

【０１９０】
表４から明らかな通り、ＺＧ１００１のｍＲＮＡは膵臓、脾臓、および脳で発現し、乳癌で発現が増加した。
この結果より、上記ｃＤＮＡおよび該ｃＤＮＡによってコードされるタンパク質は、癌やアルツハイマー病などの治療や診断に応用できる。また該ｃＤＮＡによってコードされるタンパク質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性がある。
【０１９１】
実施例１１ 各完全長ｃＤＮＡがコードするタンパク質の総合解析
（１）ｄｎａｆｏｒｍ３４８３７（配列番号１、１４）
ｄｎａｆｏｒｍ３４８３７のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、膵臓および副精巣由来脂肪細胞で強く発現することが分かった。さらに、タンパク質−タンパク質相互作用解析では、該ｃＤＮＡにコードされるタンパク質が核内ステロイド受容体の一つであるＰＸＲ２と相互作用することが確認された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核に存在し、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０１９２】
（２）ｄｎａｆｏｒｍ６３１６６（配列番号２、１５）
ｄｎａｆｏｒｍ６３１６６のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、延髄および乳腺で強く発現することが分かった。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核に存在し、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる
【０１９３】
（３）ｄｎａｆｏｒｍ３３３８３（配列番号３、１６）
ｄｎａｆｏｒｍ３３３８３のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。前記実施例４に記載したＰＦＡＭ検索の結果によると、本タンパク質はＣ末端に６個のＣ２Ｈ２ ｔｙｐｅ Ｚｉｎｃ Ｆｉｎｇｅｒ構造をもち、Ｎ末端側に蛋白相互作用にかかわるＳＣＡＮ ｄｏｍａｉｎ， ＫＲＡＢ ｄｏｍａｉｎをもつ蛋白質である。また、ＰｒｅＬｏｃによる蛋白質細胞内局在予測によると、核局在が強く示唆される。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想される多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。従って、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、ｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる
【０１９４】
（４）ｄｎａｆｏｒｍ３９５３０（配列番号４、１７）
ｄｎａｆｏｒｍ３９５３０のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることを示された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、ｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０１９５】
（５）ｄｎａｆｏｒｍ３８８６１（配列番号５、１８）
ｄｎａｆｏｒｍ３８８６１のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、全身で強く発現し、特に膵臓、肺で強力に発現することが分かった。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０１９６】
（６）ｄｎａｆｏｒｍ６０４４１（配列番号６、１９）
ｄｎａｆｏｒｍ６０４４１のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、全身で強く発現し、特に膵臓、肺で強力に発現することが分かった。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０１９７】
（７）ｄｎａｆｏｒｍ４２５１５（配列番号７、２０）
ｄｎａｆｏｒｍ４２５１５のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、脳に特異的に発現し、特に前頭様皮質で強力に発現することが分かった。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌や神経疾患などの治療や診断に応用できる。
【０１９８】
（８）ｄｎａｆｏｒｍ４１１４３（配列番号８、２１）
ｄｎａｆｏｒｍ４１１４３のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、全身で発現するが、膵臓、肺、脂肪で比較的強く発現した。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０１９９】
（９）ｄｎａｆｏｒｍ３４１９６（配列番号９、２２）
ｄｎａｆｏｒｍ３４１９６のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、ｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０２００】
（１０）ｄｎａｆｏｒｍ３７４７９（配列番号１０、２３）
ｄｎａｆｏｒｍ３７４７９のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、ｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる
【０２０１】
（１１）ｄｎａｆｏｒｍ５９６３５（配列番号１１、２４）
ｄｎａｆｏｒｍ５９６３５のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、ｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌などの治療や診断に応用できる。
【０２０２】
（１２）ｄｎａｆｏｒｍ３３７７３（配列番号１２、２５）
ｄｎａｆｏｒｍ３３７７３のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、全身で強く発現するが、特に肺、膵臓、脂肪、脳で強力に発現することが分かった。さらに、タンパク質−タンパク質相互作用解析では、該ｃＤＮＡにコードされるタンパク質が糖尿病の原因となりうるＳＲＰ４０と相互作用することも確認された。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌および神経疾患などの治療や診断に応用できる。
【０２０３】
（１３）ｄｎａｆｏｒｍ５１２１８（配列番号１３、２６）
ｄｎａｆｏｒｍ５１２１８のｃＤＮＡにコードされるタンパク質は、前記実施例９に記載のＳＰＲ測定装置（ＢＩＡＣＯＲＥ社製）を用いた５３種の標的配列を有するＤＮＡに対する結合実験の結果、該標的配列の少なくとも１つに結合し、該蛋白質が結合した標的配列に選択性を持ったＤＮＡ結合蛋白質であることが示された。また、発現解析からは、全身で強く発現するが、特に肺、膵臓、脂肪、脳で強力に発現することが分かった。
前記実施例４に記載の解析およびこれらの実験結果から、この蛋白質は核での存在が予想され、多量体あるいは他の蛋白質との相互作用をする可能性のあるＤＮＡ結合蛋白質であると考えられる。したがって、該ｃＤＮＡおよび該ｃＤＮＡによってコードされる蛋白質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があり、たとえば糖尿病や癌および神経疾患などの治療や診断に応用できる。
【０２０４】
【発明の効果】
本発明のタンパク質およびそれをコードするＤＮＡはＤＮＡ結合活性等を有することから、該タンパク質あるいはそれをコードするＤＮＡを用いて該活性を調節する物質をスクリーニングすることができ、該タンパク質が関連する疾患等に作用し得る医薬の開発に有用である。
本出願は、２００２年４月１９日付けの日本特許出願（特願２００２−１１７８４０）、２００２年４月３０日付けの日本特許出願（特願２００２−１２８４１８）、および２００２年１２月４日付けの日本特許出願（特願２００２−３５２４６９）に基づくものであり、その内容はここに参照として取り込まれる。また、本明細書にて引用した文献の内容もここに参照として取り込まれる。
【０２０５】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and a transgenic cell into which the DNA has been introduced. And antibodies that specifically bind to the protein.
[0002]
[Prior art]
Obtaining cDNA and analyzing its base sequence are indispensable for analyzing the physiological activity of a protein expressed in a living body and developing a method for utilizing the protein based on the activity. Furthermore, creating a library in which full-length cDNAs corresponding to all gene types are cataloged is one of the important issues of the human genome project. The cataloged library means that there is no duplication in the cDNAs contained in the library, and refers to a library containing one type of each cDNA.
[0003]
The full-length cDNA cloning method is described in JP-A-9-248187 and JP-A-10-127291. This method comprises the steps of binding a molecule serving as a tag to a diol structure present at the 5 'cap site of mRNA, using the mRNA bound with the tag molecule as a template, oligo dT as a primer, and reverse transcription to an RNA-DNA complex. And separating the complex having a DNA corresponding to the full length of the mRNA using the function of the tag molecule.
[0004]
As an efficient reverse transcription method, a method for performing the transcription at a high temperature such that the template does not form a higher-order structure has been developed (Japanese Patent Laid-Open No. Hei 10-84661). Furthermore, a cloning vector has been developed which can uniformly clone a DNA fragment contained in a synthesized full-length cDNA library regardless of its chain length (Japanese Patent Application Laid-Open No. H11-9273).
[0005]
A full-length cDNA library produced by such a technique does not necessarily include all the elements that are different evenly as individual elements of the library, and is present only in clones with a high abundance ratio or, conversely, in very small amounts. There are clones. Since a clone existing only in such a trace amount is highly likely to be novel, a subtraction method and a normalization method for enriching such a clone have also been developed (Japanese Patent Application Laid-Open No. 2000-325080; Carnini, P. et al. et al., Genomics, 37, 327-336 (1996)).
[0006]
The nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained can be identified by a known method, but the physiological activity of the protein encoded by the cDNA is still unknown. Remains.
[0007]
[Problems to be solved by the invention]
The present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, identifies the biological activity of the protein encoded by the cDNA sequence and determines the biological activity. It is an object of the present invention to propose a method of using a protein based thereon and DNA encoding the same.
[0008]
[Means for Solving the Problems]
The present inventors analyzed the nucleotide sequence of a cDNA clone in a mouse full-length cDNA library, and searched a database based on the homology of the sequence. The sequence was found and the proteins encoded by these cDNAs were identified as having DNA binding activity. Furthermore, the expression levels of these cDNAs in each tissue and the proteins encoded by the cDNAs were actually obtained and analyzed for their interaction. In addition, the DNA binding activity or transcription control activity of the proteins encoded by these cDNAs was analyzed. Furthermore, human homolog DNA was obtained based on the nucleotide sequence of the cDNA, and the expression level of the obtained human homolog DNA in each tissue was analyzed. The present invention has been achieved based on these findings.
[0009]
That is, according to the present invention, the following inventions (1) to (15) are provided.
(1) The following protein of (a) or (b):
(A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 14 to 26 or 36;
(B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of any of SEQ ID NOs: 14 to 26 or 36, and which has DNA binding activity.
[0010]
(2) DNA encoding the protein of (1).
(3) A full-length cDNA encoding the protein according to (1).
(4) Any one of the following DNAs (a), (b) or (c):
(A) DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 13 or 35.
(B) a protein having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence set forth in any one of SEQ ID NOs: 1 to 13 or 35, and having a DNA binding activity DNA encoding.
(C) having a base sequence capable of hybridizing under stringent conditions to a DNA having the base sequence of any one of SEQ ID NOs: 1 to 13 or 35 or a sequence complementary thereto, and having a DNA binding activity DNA encoding a protein.
[0011]
(5) A recombinant vector containing the DNA according to any one of (2) to (4).
(6) A gene-introduced cell into which the DNA according to any of (2) to (4) or the recombinant vector according to (5) is introduced, or an individual comprising the cell.
(7) The protein according to (1), which is produced by the cell according to (6).
[0012]
(8) A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any of (2) to (4), and an antisense having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of a sense oligonucleotide and an oligonucleotide derivative of the sense or antisense oligonucleotide.
[0013]
(9) An antibody or a partial fragment thereof that specifically binds to the protein of (1) or (7).
(10) The antibody according to the above (9), wherein the antibody is a monoclonal antibody.
(11) The antibody according to (10), wherein the monoclonal antibody has an action of neutralizing the DNA binding activity of the protein according to (1) or (7).
[0014]
(12) An activity-regulating substance for a protein according to (1) or (7), wherein the substance is brought into contact with a test substance and a change in the activity of the protein caused by the test substance is measured. Screening method.
(13) A method for regulating the expression of a DNA, wherein the test substance is brought into contact with the gene-introduced cell according to (6) and a change in the expression level of the DNA introduced into the cell is detected. Screening method.
(14) at least one amino acid sequence information selected from the amino acid sequence of the protein described in the above (1) and / or selected from the DNA base sequence described in any of the above (2) to (4) A computer-readable recording medium storing at least one or more base sequence information.
(15) A carrier to which the protein according to (1) and / or the DNA according to any of (2) to (4) are bound.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail.
(1) Acquisition of full-length cDNA and analysis of nucleotide sequence
The DNA of the present invention is a protein consisting of the amino acid sequence of SEQ ID NOS: 14 to 26 or 36, or one or several amino acids in the amino acid sequence (the number is not particularly limited; Or less, preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less) amino acid residue substitution, deletion, insertion, addition, or inversion, and Any substance can be used as long as it can encode a protein having DNA binding activity. Specifically, it may be only the translation region encoding the amino acid sequence or may include the full length of the cDNA.
[0016]
Specifically, examples of the DNA containing the full-length cDNA include a DNA having the nucleotide sequence of SEQ ID NOS: 1 to 13 or 35, and the like. In addition, as the translation regions, base numbers 134 to 1690 of SEQ ID NO: 1, base numbers 1751 to 2197 of SEQ ID NO: 2, base numbers 662 to 3259 of SEQ ID NO: 3, base numbers 661 to 1476 of SEQ ID NO: 4, SEQ ID NO: 4 Nucleotide Nos. 213 to 1703 of No. 5, Nucleotide Nos. 122 to 1612 of SEQ ID No. 6, 156 to 560 of SEQ ID No. 7, 149 to 1591 of SEQ ID No. 8, 597 to 1982 of SEQ ID No. 9, SEQ ID NO. SEQ ID NOs: 230 to 907, 566 to 1228 of SEQ ID NO: 11, 487 to 2127 of SEQ ID NO: 12, 167 to 2950 of SEQ ID NO: 13, and 1 to 2904 of SEQ ID NO: 35 And those having a sequence. Further, the DNA of the present invention includes not only the full length of the above-mentioned cDNA but also those containing the above-mentioned translation region and a portion adjacent to the 3 'and / or 5' end thereof, which is the minimum necessary for the expression of the translation region. .
[0017]
The DNA of the present invention may be obtained by any method as long as it can be obtained, but specifically, for example, can be obtained by the method described below. First, mRNA is prepared from a suitable animal, preferably a mammalian tissue or the like by a method known per se and generally used. Next, cDNA is synthesized using this mRNA as a template. At this time, a 5 ′ cap (^7MeG_pppN) A molecule serving as a tag is chemically bonded to a diol structure specific to a site, and reverse transcription is performed using this mRNA as a template and oligo dT as a primer. Then, only the full-length cDNA is separated using the function of the tag molecule. It is preferable to use the method (JP-A-9-248187; JP-A-10-127291). In addition, in the case of reverse transcription, in order to prevent the template from forming a higher-order structure and lowering the efficiency of reverse transcription, in the presence of trehalose or the like, use a thermostable reverse transcriptase at a high temperature. It is preferable to use a method of performing reverse transfer (Japanese Patent Laid-Open No. 10-84661). Here, high temperature means 40-80 degreeC.
[0018]
The thus obtained cDNA is cloned by inserting it into an appropriate cloning vector. The vector used herein has a recombination recognition sequence at both ends of a cloning site capable of uniformly cloning DNAs of various chain lengths, and is a linear vector inserted into a host by a method other than infection. (JP-A-11-9273) is preferably used. In the cDNA library thus obtained, not all clones exist uniformly (hereinafter, this may be referred to as "cataloged"), but only a very small amount exists in this library. A clone that does not have a high probability of being new. Therefore, it is preferable to use a subtraction method or a normalization method for enriching such clones (Japanese Patent Laid-Open No. 2000-325080; Carinci, P. et al., Genomics, 37, 327-336 (1996)).
[0019]
The nucleotide sequence of the cataloged cDNA library is analyzed by a commonly used method known per se. In the case of the full-length cDNA, the DNA of the present invention is obtained by converting the base sequence obtained from the sequence based on the terminal 100 into BLAST (http://www.ncbi.nlm.nl) using NCBI databases such as GenBank, EMBL, DDBJ, and PDB. nih.gov/BLAST/; National Center of Biotechnology Information), the sequence showing the highest homology has a homology of 30% or less and the highest homology with respect to the full length of the translation region of the DNA. Even the sequences shown whose homology is 40% or less are newly subjected to the following analysis.
[0020]
Examples of the DNA having the nucleotide sequence of such a full-length cDNA include those having the nucleotide sequence of SEQ ID NOS: 1 to 13 or 35. In addition, as the translation regions, base numbers 134 to 1690 of SEQ ID NO: 1, base numbers 1751 to 2197 of SEQ ID NO: 2, base numbers 662 to 3259 of SEQ ID NO: 3, base numbers 661 to 1476 of SEQ ID NO: 4, SEQ ID NO: 4 Nucleotide Nos. 213 to 1703 of No. 5, Nucleotide Nos. 122 to 1612 of SEQ ID No. 6, 156 to 560 of SEQ ID No. 7, 149 to 1591 of SEQ ID No. 8, 597 to 1982 of SEQ ID No. 9, SEQ ID NO. SEQ ID NOs: 230 to 907, 566 to 1228 of SEQ ID NO: 11, 487 to 2127 of SEQ ID NO: 12, 167 to 2950 of SEQ ID NO: 13, and 1 to 2904 of SEQ ID NO: 35 And those having the sequence shown in the sequence.
[0021]
The thus obtained novel nucleotide sequence was subjected to homology search by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)). protein feature search by HPMMPFAM, which is one of the functional groups of homology search and HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14, 755-763 (1998)). //Pfam.wustl.edu) or the like, the function of the protein encoded by the nucleotide sequence can be estimated.
[0022]
In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various kinds of annotation information associated with hit sequences having sufficiently significant homology obtained as a result of the search. Here, a sufficiently significant hit sequence means that the identity between the catalytic domain portion of the registered sequence and the corresponding portion of the DNA of the present invention is 10 as an e-value.^-4Show the following or 30% or more.
[0023]
For example, if many of the catalytic domain sequences hit in the top rank have been confirmed to function as DNA binding proteins, clones to be analyzed that are similar in sequence to those also have the same function, that is, DNA binding activity. The expectation holds.
[0024]
In HMMPFAM, analysis is performed by a method of checking whether or not a base sequence to be analyzed has a feature of a base sequence of an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and even if a function cannot be clarified by comparing the full length of one sequence to one sequence, if there is a characteristic region in the sequence, it can be found and its function can be predicted. . A specific example of the function prediction of the protein thus performed will be described below.
[0025]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 was obtained by BLAST search using RIKEN full-length enriched library, clone: 9530049C15, SCAN domain containing protein, and e-value: 5 × 10^-171, 100% homology, and Zinc finger protein 192 (LD5-1) is a Kruppel gene family with SCAN box domain, but e-value: 2 × 10⁻⁹⁴, 42% homology with Homo sapiens zinc finger protein (ZFP) and e-value: 4 × 10⁻⁹³, With 41% homology. From these results, it can be inferred that the protein encoded by the nucleotide sequence of SEQ ID NO: 1 is Zinc finger protein.
In the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1, a protein characteristic search by HMMPFAM finds seven times a sequence (a nucleotide sequence entered as LIM in Pfam) exhibiting ZF-C2H2 characteristics.
From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 is a zinc finger type transcription factor.
[0026]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 was obtained by BLAST search from RIKEN cDNA 2810411K16 and e-value: 2 × 10^-76100% homology over 139 amino acids, RIKEN full-length enriched library, clone: 2810411K16: SCAN domain containing protein, and e-value: 6 × 10^-76And 99% homology over 139 amino acids with Homo sapiens cDNA FLJ14478 fis, clone MAMMA1001633, e-value: 1 × 10⁻⁶¹, With 86% homology over 140 amino acids.
In addition, the SCAN sequence was found at base numbers 1880 to 2167 by protein characteristic search using HMMPFAM. From these facts, it can be inferred that the protein encoded by the base sequence shown in SEQ ID NO: 2 is a DNA-binding protein.
[0027]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3 was determined by BLAST search as Homo sapiens, clone MGC: 13310 IMAGE: 4110431 and e-value: 5 × 10^-145, 40% homology over 919 amino acids, and Zinc finger protein 180 (HHZ168), e-value: 2 × 10⁻⁶³, 51% homology over 231 amino acids, further with Rattus norvegicus Cys2 / His2 zinc finger protein (rKr1) and e-value: 6 × 10⁻⁶³, With 56% homology over 192 amino acids.
In addition, a protein characteristic search by HMMPFAM found six places of zf-C2H2 at base numbers 786 to 3275, and from these characteristics, it can be inferred that the protein encoded by the base sequence described in SEQ ID NO: 3 is a DNA-binding protein. .
[0028]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 was obtained by BLAST search using Macaca fascicularis brain cDNA, clone: QccE-18103. And e-value: 2 × 10^-95, With 79% homology over 220 amino acids, and with Homo sapiens putative transcription factor CR53, e-value: 1 × 10⁻³², 56% homology, and further homologous to Homo sapiens, Simulator to zinc finger protein 202, clone MGC: 15660 IMAGE: 3347511 and e-value: 1 × 10⁻³², With 56% homology over 134 amino acids.
In addition, when a protein characteristic search is performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 by using HMMPFAM, a SCAN sequence (a nucleotide sequence that is entered as SCAN in Pfam) is found.
From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 4 is a transcription factor.
[0029]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5 was obtained by BLAST search using Mus musculus, zinc finger proliferation 1, and e-value: 6 × 10⁻⁹⁷, 41% homology over 491 amino acids with mouse Zinc finger protein 38 (Zfp-38) (CtFIN51) (Transscription factor RU49) and e-value: 2 × 10^-96, 41% homology over 491 amino acids, as well as Hypothetical zinc finger protein KIAA0426, e-value: 2 × 10^-95With 44% homology over 532 amino acids.
Further, zf-C2H2 was found eight times at base numbers 929 to 1585 by protein characteristic search using HMMPFAM, and from these characteristics, it is presumed that the protein encoded by the base sequence of SEQ ID NO: 5 is a DNA-binding protein. it can.
[0030]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6 was determined by BLAST search to be Mus musculus, zinc finger proferation 1, clone MGC: 18498 IMAGE: 3981599, and e-value: 6 × 10⁻⁹⁷, 41% homology over 491 amino acids, and e-value: 2 × 10 with mouse Zinc finger protein 38 (Zfp-38) (CtFIN51) (Transscription factor RU49).^-96, 41% homology over 491 amino acids, as well as Hypothetical zinc finger protein KIAA0426, e-value: 2 × 10^-95With 44% homology over 532 amino acids.
Further, zf-C2H2 was found eight times at base numbers 929 to 1585 by protein characteristic search using HMMPFAM. From these characteristics, it can be inferred that the protein encoded by the base sequence of SEQ ID NO: 6 is a DNA-binding protein. .
[0031]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 7 was analyzed by BLAST search with a STATc transcriptional repressor and an e-value: 4 × 10^-28With 53% homology over 127 amino acids, and a forkhead-related transcription factor 2 and e-value: 4 × 10^-23With a homology of 58% over 109 amino acids, and with Transcription regulatory protein SNF5, e-value: 2 × 10⁻²⁴And has 61% homology over 118 amino acids.
STATc transcriptional repressor is based on the literature information (Mol. Cell, 7 (4), 779-88 (2001)) in the database and relates to the control of the initial development rate and the timing of terminal differentiation, and the ecmA gene. Can function as a repressor that regulates the expression of, and forkhead-related transcription description 2 can be expressed in the lung and placenta from literature information (J. Biol. Chem. 1998, 273 (36): 23335-43) in a database. Binding of the cis factor of some lung-specific genes with the expressed transcription factor, and in addition Transcription regulatory protein SNF5, is a statement in the database. Information (Mol Cell Biol, 1990, 10 (11):... 5616-25) seen each be involved in transcriptional regulation of genes regulated by glucose and phosphate from.
These characteristics suggest that the protein encoded by the nucleotide sequence of SEQ ID NO: 7 is a transcription factor.
[0032]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 8 was analyzed by BLAST search to find the zinc finger protein 151 (pHZ-67) and e-value: 1 × 10^-25At 48% homology over 121 amino acids, and with Myc-interacting zinc finger protein, e-value: 2 × 10^-25With 48% homology over 121 amino acids. From these results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 8 is a Zinc finger-type DNA binding protein.
The Myc-interacting zinc finger protein is known to be Myc-interacting zinc finger protein from literature information (Curr Top Microbiol Immunol 1997; 224: 137-46) in the database.
In addition, a protein characteristic search using HMMPFAM finds a sequence (an amino acid sequence that is entered as BTB in Pfam) showing characteristics relating to protein dimerization in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 8. In addition, one Zinc finger domain (an amino acid sequence entered as zf-C2H2 in Pfam) is also found.
From these characteristics, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 8 is a Zinc finger type DNA binding protein.
[0033]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 was obtained by BLAST search using Hypothetical zinc finger protein KIAA1473 and e-value: 1 × 10⁻⁵⁹With a homology of 37% over 295 amino acids, and Zinc finger protein 135 and e-value: 2 × 10^-58With 39% homology over 277 amino acids. From these results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9 is a Zinc finger type DNA binding protein.
The above-mentioned Zinc finger protein 135 protein is known to be a zinc finger Kruppel family from literature information (Genomics 1995 May 20; 27 (2): 259-64) in a database, and it is known that the protein is a developmental disorder. We understand that we are involved.
In addition, a protein feature search using HMMPFAM reveals that Zinc finger domain (an amino acid sequence entered as zf-C2H2 in Pfam) is found at nine positions in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9.
From these characteristics, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 9 is a Zinc finger type DNA binding protein.
[0034]
As a result of a BLAST search, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 was found to be PR-domain zinc finger protein 5 and e-value: 3 × 10^-13And a homology of 50% over 62 amino acids, and an e-value: 2 × 10 5 with the zinc finger protein with interaction domain.^-12And has 43% homology over 81 amino acids. From these results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 is a Zinc finger-type DNA binding protein.
Further, the above-mentioned zinc finger protein with interaction domain protein is known to be a zinc finger protein with interaction from literature information (Genes Dev 1994 Jul 15: 8 (14): 1664-77) in the database.
In addition, a protein feature search using HMMPFAM finds two Zinc finger domain (an amino acid sequence that is entered as Pfam as zf-C2H2) in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10.
From these characteristics, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 10 is a Zinc finger type DNA binding protein.
[0035]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 11 was obtained by BLAST search using PR-domain zinc finger protein 5 and e-value: 4 × 10^-13And 50% homology over 62 amino acids, and e-value: 3 × 10 5 with zinc finger protein with interaction domain.^-11And has 41% homology over 81 amino acids. From these results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11 is a Zinc finger type DNA binding protein.
Further, when a protein characteristic search was performed by HMMPFAM, Zinc finger domain (an amino acid sequence entered as zf-C2H2 in Pfam) was found in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11.
From these characteristics, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 11 is a Zinc finger type DNA binding protein.
[0036]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 12 was obtained by BLAST search using Mus musculus zinc finger transcription factor Kaiso and e-value: 2 × 10⁻²⁷With a homology of 58% over 94 amino acids, and a Xenopus laevis BTB / POZ zinc finger transscription factor XKaiso with e-value: 2 × 10⁻²⁹And has 48% homology over 136 amino acids.
In addition, by a protein characteristic search using HMMPFAM, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12 shows a sequence showing the characteristics of BTB (for BR-C, ttk and bab) or POZ (for Pox virus and Zinc finger) domain. (An amino acid sequence entered as BTB in Pfam), a sequence exhibiting characteristics of Msx-interacting-zinc finger (an amino acid sequence entered as zf-MIZ in Pfam), and a sequence exhibiting characteristics of Zinc finger, C2H2 type (Pfam amino acid sequence entered as zf-C2H2).
From these characteristics, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 is a Kaiso-like transcription factor having a zinc finger.
[0037]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 13 was analyzed by BLAST search with Xenopus laevis DNA-methylation dependent transcriptional repressor Kaiso-like protein (Kaiso) and e-value: 4 × 10⁻³²And 48% homology over 137 amino acids, and e-value: 4 × 10 with Xenopus laevis BTB / POZ zinc finger transcription factor XKaiso.⁻³²With 48% homology over 137 amino acids.
In addition, by a protein characteristic search using HMMPFAM, the amino acid sequence encoded by the base sequence described in SEQ ID NO: 13 shows a sequence (Pfam) showing the characteristics of the BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain. An amino acid sequence entered as BTB), a sequence exhibiting characteristics of Msx-interacting-zinc finger (an amino acid sequence entered as zf-MIZ in Pfam), and a sequence exhibiting characteristics of Zinc finger, C2H2 type (zf-in Pfam). Amino acid sequence entered as C2H2).
From these characteristics, it can be inferred that the nucleotide sequence shown in SEQ ID NO: 13 is a part of the sequence of a gene for a transcription factor of Kaiso-like having zinc finger.
[0038]
The DNA of the present invention thus obtained, whose base sequence is determined, and whose function is estimated, has the base sequence of SEQ ID NOS: 1 to 13 or 35 or the base sequence shown above as its translation region. In addition, in these base sequences, one or several (the number is not particularly limited, but is, for example, 60 or less, preferably 30 or less, more preferably 20 or less, still more preferably 10 or less). Or less, particularly preferably 5 or less), a DNA encoding a protein having a base sequence with deletion, substitution, and / or addition of DNA, and having a DNA binding activity; DNA that hybridizes under gentle conditions and encodes a protein having DNA binding activity is also included. As described above, these DNAs comprise an amino acid sequence in which one or several amino acid sequences have been deleted, substituted and / or added in the amino acid sequence of the protein shown in SEQ ID NO: 14 to 26 or 36, and have a DNA binding activity. And those encoding proteins having the formula:
[0039]
Here, the DNA that hybridizes under stringent conditions refers to the nucleotide sequence shown in SEQ ID NOS: 1 to 13 or 35 or its complementary sequence in a BLAST analysis of 80% or more, preferably 90% or more, and more preferably 95% or more. DNAs containing the base sequence having the above homology are exemplified. Further, the hybridization under stringent conditions means that the reaction is carried out in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, and a salt concentration of 15 mM to 300 mM, preferably The washing can be performed according to a method of washing in a washing solution of 15 mM to 60 mM or the like.
[0040]
Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).
[0041]
The nucleotide sequences described in SEQ ID NOs: 1 to 13 are derived from mouse. A human cDNA library was prepared according to the above-described method for preparing a cDNA library, and the library was subjected to SEQ ID NO: By performing hybridization using a DNA fragment having the nucleotide sequence of 1 to 13 as a probe, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NOS: 1 to 13 can also be obtained. . DNAs that hybridize under stringent conditions to DNAs having the nucleotide sequences of SEQ ID NOs: 1 to 13 or their complementary sequences also include DNAs encoding such human homologs.
[0042]
Further, it is also possible to predict the base sequence of the human homolog DNA by using informatics, and to obtain the human homolog DNA from the above-mentioned human cDNA library or the like based on the base sequence.
In general, as a method for predicting a base sequence encoding a homolog protein of a target protein using informatics, for example, (i) using a base sequence of a target cDNA as a query, A method of performing homology search using a BLAST or the like on a database (including a cDNA database predicted by informatics), or (ii) using a BLAST or the like on a human or other EST database using a target cDNA as a query A method in which the sequence of the hit EST is linked to the base sequence of the target cDNA by referring to the base sequence of the target cDNA, and (iii) the base sequence of the target cDNA is used as a query in a genome database such as human. Perform a homology search using BLAST, etc. The position on the genome where the cDNA gene is located is specified, and Genscan (http://genes.mit.edu/GENSCAN.html) or Sim4 (Genome Res., 8: 976-74) is determined for the genomic region. 1998)) and the like, and a method of predicting the nucleotide sequence of the gene portion in the genome.
[0043]
When predicting the nucleotide sequence of human homolog DNA of mouse-derived cDNA, any of the above methods can be used, but any cDNA having the nucleotide sequence of SEQ ID NOS: 1 to 13 of the present invention is novel, In the above method (i), it is considered that the nucleotide sequence of the human homolog DNA cannot be obtained, so the method described in (ii) or (iii) is preferably used.
[0044]
Based on the predicted nucleotide sequence of the human homolog DNA, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NOS: 1 to 13 can be obtained from the above human cDNA library. . As a specific acquisition method, for example, using a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 ′ end and 3 ′ end of the predicted human homolog DNA, PCR is performed using the above human cDNA library as a template. And a method of performing hybridization with the above human cDNA library using a partial sequence of the predicted human homolog DNA as a probe.
[0045]
Generally, a similar gene having a nucleotide sequence having a high homology with the nucleotide sequence of the target gene is referred to as a “homolog”, and the above-described method also aims to obtain a human homolog. It is important to confirm that not only the similarity but also that the gene obtained as a homolog is a family member of the target gene. Genes acquired as "homologs" between two species of organisms are likely to be "orthologs", the same gene evolved from a common ancestral gene, and differ from each other caused by duplication from a common ancestral gene It may be a "paralog" that is a gene.
[0046]
That is, in order for the human-derived DNA obtained as a homologue to have the same function as the protein of the present invention, the function of the protein encoded by the human-derived DNA must be In order to verify the function of the protein of the present invention as a mouse, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
[0047]
For example, the following method is used as a method for confirming the ortholog.
(1) First, the homology between the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA is analyzed. Next, using the base sequence of the cDNA of the present invention as a query, a homology search was performed for human base sequences contained in international base sequence databases such as DDBJ, EMBL, and GenBank, and patent databases. Confirm that the degree of matching of the base sequence is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. Further, (2) homology is analyzed between the obtained nucleotide sequence of the human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the base sequence of the obtained human homolog DNA as a query, a homology search was performed for the mouse base sequence contained in the international base sequence database such as DDBJ, EMBL, and GenBank, and in the patent database. Confirm that the degree of matching of the base sequence is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. By confirming the above (1) and (2), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (1) and (2) above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree and examining it. In addition, it is preferable that the degree of coincidence by the homology analysis described in the above (1) and (2) be analyzed as the degree of coincidence over the entire length of the query.
[0048]
By performing a homology search by BLAST or a protein feature search by HMMPFAM on the nucleotide sequence of the thus obtained human homologous DNA or orthologous DNA, the function of the protein encoded by the nucleotide sequence can be estimated and confirmed. The human ortholog of the cDNA of the present invention includes, for example, a DNA having the base sequence of SEQ ID NO: 35, and the human ortholog of the protein of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 36, and the like. Can be
DNAs that hybridize under stringent conditions to DNAs having the nucleotide sequences of SEQ ID NOs: 1 to 13 or their complementary sequences include DNAs encoding such human homologues or orthologous proteins.
[0049]
(2) Protein encoded by the novel cDNA
The translation region of the protein encoded by the DNA of the present invention is, for example, a base sequence of the DNA which is converted into amino acids by three types of reading frames, and the range encoding the longest polypeptide is defined as the translation region of the present invention. The amino acid sequence can be determined. Examples of such an amino acid sequence include those described in SEQ ID NOs: 14 to 26 or 36. The protein of the present invention is not limited to the above-mentioned amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted and / or added, and has a DNA binding activity. And those having the following.
[0050]
As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in the above (1) by an appropriate method is preferably used. Specifically, a recombinant vector inserted into a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and this recombinant vector is used to transform a suitable host microorganism or introduced into a suitable cultured cell. Thus, it can be obtained by purifying this.
[0051]
When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in a salt form, it can be converted to a free form or another salt. can do. Such salts of the protein of the present invention are also included in the protein of the present invention. Further, a protein produced by the transformant may be modified before or after purification by applying an appropriate protein modifying enzyme to modify the protein arbitrarily or partially removing the polypeptide. Can be. These modified proteins are also included in the scope of the present invention as long as they have the above-mentioned DNA binding activity.
[0052]
When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. Any of vectors may be used. Of these, usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3 and pET11 (manufactured by Stratagene) and pGEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and pESP- when the host is yeast. I expression vector (Stratagene) and the like. In the case of insect cells, BacPAK6 (Clontech) and the like are used. When the host is an animal cell, ZAP Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech) and the like can be mentioned.
[0053]
When using a vector into which the expression control region has not been inserted, it is necessary to insert at least a promoter as the expression control region. Here, as the promoter, a promoter contained in a host microorganism or a cultured cell can be used. However, the promoter is not limited thereto. For example, when the host is Escherichia coli, T3, T7, tac, A lac promoter or the like can be used. In the case of yeast, an nmt1 promoter, a Gal1 promoter, or the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used.
[0054]
When a host capable of functioning with a mammalian-derived promoter is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be performed by linking the DNA or a DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector.
[0055]
The recombinant vector thus prepared can be used to transform a host described below by a method known per se to prepare a DNA transductant. As a method for introducing the vector into a host, specifically, a heat shock method (J. Mol. Biol., 53, 154 (1970)), a calcium phosphate method (Science, 221, 551, (1983)), DEAE Dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984)). )), And the electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
[0056]
The host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cells, or Animal cells and the like can be mentioned. Specifically, BL21 and XL-2Blue (manufactured by Stratagene) and the like for Escherichia coli, SP-Q01 (manufactured by Stratagene) and the like for yeast, and AcNPV (J. Biol. Chem., 263, 7406, and the like) for baculovirus. 1988)) and its host Sf-9 (J. Biol. Chem., 263, 7406, (1988)). Examples of animal cells include mouse fibroblast C127 (J. Viol., 26, 291, (1978)) and Chinese hamster ovary cell CHO cells (Proc. Natl. Acad. Sci. USA, 77, 4216, (1980)). Of these, African green monkey kidney-derived COS-7 (ATCC CRL1651: American Type Culture Collection-preserved cell) is preferably used because of its expression level and simplicity of screening.
[0057]
In addition to the above-described expression method using a protein expression vector, a homologous recombination technique (AA Vertes et al., Biosci) in which a DNA fragment of the present invention linked to a promoter is directly inserted into a chromosome of a host microorganism. Biotechnol. Biochem., 57, 2036 (1993)) or a transposon or an insertion sequence (A.A. Vertes et al., Molecular Microbiol., 11, 739, (1994)). It can also be made.
[0058]
The obtained culture is obtained by collecting cells or cells by a method such as centrifugation, suspending the cells or cells in a suitable buffer, and sonicating, lysozyme, and / or freezing and thawing. After the disruption, a crude protein solution is obtained by centrifugation, filtration, or the like, and further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained. In addition to the above-described expression method using the protein expression recombinant vector, protein expression is induced by subjecting the DNA of the present invention obtained in (1) to a cell-free transcription / translation system to obtain the protein of the present invention. be able to. The cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription from DNA to mRNA and translation from mRNA to protein. Refers to any system in which the protein being synthesized is synthesized. Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell and a bacterial cell, or an extract from a part thereof. A transcription / translation system prepared based on an extract from Erythrocytes, wheat germ and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
[0059]
Separation and purification of the protein of the present invention from the obtained transcription / translation product of the cell-free transcription / translation system can be carried out by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, glutathione-S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated, and expressed as described above. The protein can be purified by utilizing the affinity of the protein with a substance having affinity.
[0060]
The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Brilliant Blue (manufactured by Sigma) or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method of performing. In general, it is known that an expressed protein is cleaved (processed) by a proteolytic enzyme present in a living body. The protein of the present invention is naturally included in the protein of the present invention as long as it has a DNA binding activity, even if it is a partial fragment of the amino acid sequence that has been cleaved.
[0061]
By analyzing the interaction between the thus obtained protein and other proteins and DNA, it is possible to know the multifaceted functions in the living body. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal method Display method and the like can be mentioned.
[0062]
(3) Preparation of oligonucleotide and functional analysis using the oligonucleotide
Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, an antisense oligonucleotide having a partial sequence of the DNA of the present invention, a sense -An oligonucleotide such as an oligonucleotide can be prepared.
[0063]
Examples of the oligonucleotide include a DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. Specific examples include a DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence represented by any of SEQ ID NOS: 1 to 13 or 35, or a DNA having a sequence complementary to the DNA. it can. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
[0064]
In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in which a phosphoric diester bond in an oligonucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Nucleotide derivative, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, oligonucleotide derivative in which uracil in oligonucleotide is substituted with C-5 propynyluracil, uracil in oligonucleotide is Oligonucleotide derivatives substituted with C-5 thiazole uracil, oligonucleotide derivatives substituted with cytosine in the oligonucleotide with C-5 propynylcytosine, oligonucleotides Is an oligonucleotide derivative in which cytosine is substituted by phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in the oligonucleotide is substituted by 2′-O-propyl ribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose can be mentioned.
[0065]
In addition, the oligonucleotide of the present invention is prepared as a double-stranded RNA, introduced into a recipient, and inhibited by the RNA interference method for inhibiting the expression of a target gene (hereinafter referred to as the “RNAi method”). There is). As the RNA interference method, for example, a method described in (Elbashir, S., et al., Nature, 411, 494-498 (2001)) can be used. The double-stranded RNA does not necessarily have to be all RNA, and for example, those described in WO 02/10374 can be used.
[0066]
Here, the target gene may be any DNA as long as it is the DNA of the present invention. A double-stranded RNA consisting of a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is defined as a part of the base sequence of the target gene. And a sequence substantially the same as a sequence of 15 bp or more, which may be any part. Here, “substantially the same” means that it has 80% or more homology with the sequence of the target gene. The nucleotide length of the nucleotide may be any length from 15 bp to the entire length of the open reading frame (ORF) of the target gene, but a length of about 15 to 500 bp is preferably used. However, it is known that mammalian-derived cells have a signal transduction system activated in response to a long double-stranded RNA of 30 bp or more. This is called an interferon reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters a cell, PKR (dsRNA-responsive) is obtained. Protein kinase: Non-specifically inhibits the initiation of translation of many genes via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2 ', 5' oligoadenylate synthetase (Bass, B.L., Nature, 411, 428-429 (2001)), RNaseL is activated, and non-specific degradation of intracellular RNA is caused. These non-specific reactions mask the specific response of the target gene. Therefore, when a mammal, or a cell or tissue derived from the animal is used as a recipient, a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 24 bp, and most preferably 21 bp is preferably used. The double-stranded polynucleotide does not need to be entirely double-stranded, and includes those in which the 5 'or 3' end is partially protruded, but those in which the 3 'end is protruded by two bases are preferably used. The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. The single-stranded polynucleotide having self-complementarity includes, for example, one having an inverted repeat sequence.
[0067]
The method for preparing the double-stranded polynucleotide is not particularly limited, but a known chemical synthesis method is preferably used. In chemical synthesis, a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method. Specific examples of the method of association include a method in which the synthesized single-stranded polynucleotide is mixed, heated to a temperature at which the double-strand is dissociated, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing it with an appropriate enzyme.
[0068]
The transfectant into which the double-stranded polynucleotide thus prepared is introduced may be any as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species. The plant may be a monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or industry, and those that are pathogenic for plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals, including fish, cows, goats, pigs, sheep, hamsters, mice, rats, and humans, and invertebrates include nematodes and other reptiles, Drosophila melanogaster ( Drosophila), and other insects. Preferably, the cells are vertebrate cells.
[0069]
The transductant means a cell, tissue, or individual. Here, the cell may be from germline or somatic, totipotent, or pluripotent, split or undivided, parenchymal tissue or epithelium, immortalized or transformed, and the like. The cells may be gametes or embryos, in the case of embryos, single-cell or constitutive cells, or cells from multi-cell embryos, including fetal tissue. Furthermore, they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism. Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glial, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.
[0070]
As a method for introducing a double-stranded polynucleotide into a transfectant, when the transfectant is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, double-stranded polynucleotide solution Immersion, transformation, or the like. Examples of the method for introducing the gene into an embryo include microinjection, electroporation, and virus infection. When the recipient is a plant, a method of injecting or perfusing the plant into a body cavity or stromal cells, or spraying is used. In the case of an individual animal, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ocular and intraperitoneal administration. A method, an electroporation method, a virus infection, or the like is used. For methods for oral introduction, the double-stranded polynucleotide can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
[0071]
The amount of the double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced substance and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the transfectant is a human cultured cell and the double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 1000 nM is preferable.
By suppressing the expression of the gene of the present invention in the transfectant by RNA interference, the function of the protein encoded by the gene of the present invention can be confirmed, or a new function can be analyzed.
[0072]
(4) Antibodies that specifically bind to the protein of the present invention
As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. For a polypeptide used as an antigen, an epitope (antigen determination An appropriate sequence can be selected and used as the group. As a method for selecting an epitope, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
[0073]
As the polypeptide used as the above antigen, a synthetic peptide synthesized according to a known method or the protein of the present invention itself can be used. The polypeptide serving as an antigen may be prepared in an appropriate solution or the like according to a known method and immunized to a mammal, for example, a rabbit, a mouse, a rat, or the like. It is preferable to use an antigen peptide as a conjugate with a suitable carrier protein or to add an adjuvant or the like for immunization.
[0074]
The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB / c mice several times every several days to several weeks with an antigen polypeptide is used. The amount of the antigen to be taken is preferably about 0.3 to 0.5 mg / time when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.
[0075]
After immunization, blood is appropriately collected as a test, and an increase in the antibody titer is confirmed by a method such as enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting. Blood is collected from animals with increased titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specifically, for example, there is a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For purification of the antibody component, methods such as centrifugation, ion exchange chromatography, and affinity chromatography can be used.
[0076]
In addition, a monoclonal antibody can be prepared by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256, 495 (1975)). . The monoclonal antibody can be obtained, for example, by the following method.
[0077]
First, antibody-producing cells are obtained from an animal whose antibody titer has increased due to immunization with the above-described antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from the spleen, lymph nodes, peripheral blood and the like. As a myeloma to be fused with these cells, generally, a cell line obtained from a mouse, for example, a P3X63-Ag8.653 (ATCC: CRL) which is an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line is used. -1580), P3-NS1 / 1Ag4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI1640 or Iskov's modified Dulbecco's medium (IMDM) or Dulbecco's modified Eagle's medium (DMEM). It can be carried out by using a solution in which glycol (PEG) is dissolved. It can also be carried out by the electrofusion method (U. Zimmer-mann. Et al., Naturewissenschaften, 68, 577 (1981)).
[0078]
Hybridomas were prepared using a myeloma cell line that was resistant to 8-azaguanine, using 5% CO2 in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (HAT) solution.₂At 37 ° C. for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the above-described method, the hybridoma producing the antibody having a high antibody titer is separated by a limiting dilution method or the like, and the separated fused cells are cultured in an appropriate medium. The monoclonal supernatant can be obtained by purifying the resulting culture supernatant by an appropriate method such as ammonium sulfate fractionation and affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.
[0079]
When a human-derived protein is obtained as the protein of the present invention, the above-described method is applied to a Severe combined immunodeficiency (SCID) mouse transplanted with human peripheral blood lymphocytes using the polypeptide or a partial peptide thereof as an antigen. A humanized antibody can also be prepared by immunization in the same manner as described above and preparing a hybridoma of the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, DE, et al. Nature, 335, 256-259 (1988); Duchosal, MA, et al., Nature, 355, 258-262 (1992)).
[0080]
Further, RNA is extracted from the obtained hybridoma producing the human antibody, a gene encoding the target human antibody is cloned, this gene is inserted into an appropriate vector, and this is introduced into an appropriate host. By expression, human antibodies can be produced in larger quantities. Here, an antibody with low binding to an antigen can be obtained as an antibody with even higher binding by using an evolutionary engineering technique known per se. A partial fragment such as a monovalent antibody can be prepared by cleaving the Fab and Fc portions using, for example, papain or the like, and collecting the Fab portion using an affinity column or the like.
[0081]
The thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutralizing antibody that specifically binds to the protein of the present invention and thereby inhibits the DNA binding activity of the protein. There is no particular limitation on the method of selecting a substance that inhibits the activity of the protein. For example, it is possible to contact an antibody with the DNA transfectant prepared in (2) above and determine whether the function of the target protein in the transfectant is inhibited. And a method of analyzing the above.
[0082]
Such a neutralizing antibody may be used alone when the clinical application is performed, or may be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0083]
(5) Confirmation and analysis of the activity of the protein of the present invention
The protein of the present invention is prepared as a recombinant protein as described in the above (2), and by analyzing this, it can be confirmed that it has the activity estimated in the above (1). Furthermore, analysis can also be performed by combining the antibody and the like prepared as described in (4) above.
The fact that the protein of the present invention has DNA binding activity can be confirmed, for example, by contacting an appropriate double-stranded DNA with the recombinant protein and measuring the binding property of the recombinant protein to the DNA chain. it can. Specific methods include, for example, the methods described below.
[0084]
As a reaction solution, a neutral to weakly basic buffer containing 60 mM potassium chloride, 1 mM dithiothreitol, 10% glycerol, and 1 μg poly (dI-dC), for example, 20 mM Tris-HCl or HEPES buffer (pH 7 to 8) The reaction is started by adding a double-stranded DNA of an appropriate length and the recombinant protein of the present invention. After the reaction under certain conditions, the double-stranded DNA binding activity of the protein is determined by detecting the binding product of the recombinant protein and DNA.
[0085]
The binding between the recombinant protein and DNA can be detected using a method of detecting as a molecular size, a method of detecting as a difference in electric charge, a method of measuring the affinity of both, and the like.
As a method for detecting the size of a molecule, measurement by molecular sieve chromatography can be mentioned. The reaction solution is added to a column for molecular sieve chromatography equilibrated with physiological saline or 0.1 M phosphate buffer (pH 7.4) and 0.1 M HEPES (pH 7.5), and developed with the same solution. I do. Compared to the elution position of each of the recombinant protein and DNA, the bound product is eluted on the higher molecular weight side.
[0086]
Examples of the method for detecting the difference in charge include an electrophoresis method including ion exchange chromatography, gel electrophoresis, and capillary electrophoresis. In gel electrophoresis, a difference in charge and a difference in molecular size appear as a difference in mobility. The above reaction solution is electrophoresed in a polyacrylamide gel, and the gel after electrophoresis is silver-stained, Coomassie brilliant blue-stained, or fluorescent-stained. Confirmed as a band. In the case of these two methods, by labeling one end of the DNA with a fluorescent label, it is possible to detect the elution position of the DNA and the difference in mobility with higher sensitivity.
[0087]
Methods for measuring the affinity of both include affinity chromatography, surface plasmon resonance, and the like. In these methods, DNA is immobilized on a carrier, and a recombinant protein is brought into contact with the DNA, and the amount of binding is measured to determine the strength of the activity. In affinity chromatography, the bound recombinant protein is eluted with a high-concentration salt solution, denaturant, free DNA, and the like, and the amount of the eluted protein is measured. In the surface plasmon resonance method, the amount of protein bound to immobilized DNA is measured by surface plasmon resonance, and the strength of affinity can be measured based on the concentration of the bound protein.
[0088]
In addition, for proteins that bind to DNA and RNA and are involved in transcription, generally, gene transcription regulatory regions such as promoters and enhancers that are recognized and targeted by known transcription-related factors (hereinafter, referred to as “target transcription regulatory regions”). ), And a method for comprehensively analyzing transcription control activity by measuring the binding ability to the DNA and partial fragments thereof (JP-A-2001-314190). The DNA of the target transcription control region and its partial fragments used in the analysis include not only those having a known base sequence of the target transcription control region (hereinafter, this may be referred to as “target sequence”), but also known targets. The base sequence of the transcription regulatory region is analyzed and classified into a database, and each is designed. The common base sequence of the target transcription regulatory region that is commonly recognized by transcription-related factors (hereinafter, this may be referred to as a “consensus target sequence” ) May be used. Known transcription-related factors include, for example, v-jun, c-jun, junB, junD, dJRA, c-fos, fosB1, fosB2, Fra-1, LRF-1, v-maf, mafG, NF-E2 p45. , ANF-E2, fNF-E2, Nrf short form, GCN4, yAP-1, CREB-2, ATF-3, CRE-BP1, CRE-BP3, ATF-a, CREB-341, CREB-327, CREM, dCREB2 , DCREB2-b, dCREB2-c, dCREB2-d, dCREB2-q, dCREB2-r, dCREB2-s, C / EBPalpha, C / EBPbeta, p34C / EBPbeta, CHOP-10, VBP, Hlf, CPR −2, EmBP-1b, EmBP-1b, GBF1, GBF2, GBF3, CPRF-1, TAF-1, HBP-1a, GBF9, GBF1, GBF12, CPRF-3, TGA1a, TGA1b, O2, STE4, OPI1, E2A , E47, ITF-2 / SEF2-1B, SEF-1A, MyoD, p42Tal-1, HEN-1, AhR, Arnt, USF, NF-1A1, NF-1A1.1, NF-1A6, NF-1B1, NF -1B1, NF-1B2, NF-1C2 / CTF-2, CTF-4, CTF-6, RF-X1, AP2alphaA / AP-2alpha1, AP2alpha2, AP2alpha3, AP2alpha4, AP2alphaB, AP2b eta, AP2gamma, GR, AR, ER, RXR-alpha, PPARalpha, PPARgamma, COUP-TF1, HNF-4alpha1, HNF-4alpha2, CF1, GATA-1, GATA-2, GATA-3, GATA-4, AREA / NIT-2, Sp1, YY1, Egr-1, Egr-2, Egr-3, Snail, CF2-II, Evi-1, Ikaros, MZF-1, Tramtrack 69K, HOX9, CDP, HNF-1A, Nkx-2. 2, Nkx-2.5, TTF-1, Oct-1A, Oct-1B, Oct-1C, Oct-2, Oct-2.1 / Oct-2B, Pax-3, Pax-6, Pax-1, HSF1 (shor t), HSF2, dHSF, fungalHSF, c-Myb, A-Myb, v-Myb, P (long), P (short), C1 (long), C1 (short), c-Ets-1 # p54, Ets -1 # deltaV / VII, Ets-2, Elk-1, SAP-1, SAP-1b, Erg-1, p55erg, Fli-1b, E4TF1-60 / GABP-alpha, E74A, IRF-1, IRF-2 , P50, NF-ATc, NF-Atp, p91, p84, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, p53, MEF-2A, SRF, E2, TBP, SRY, Sox-5, Sox-9 -Mc, CP1A, P1B, CBF-C, AML1a, and the like are known, and the nucleotide sequences of target transcriptional regulatory regions of these transcription-related factors have also been reported (Takaaki Tamura, et al., 2nd Ed., "BioScience Experimental Medicine Supplement New. Term Library Transcription Factors ", 2nd edition, Yodosha Co., Ltd., December 1999).
[0089]
The binding between the protein having the DNA binding activity of the present invention and the DNA of the target transcription regulatory region and its partial fragments can be detected using a measurement system known per se. For example, a test sample containing the protein of the present invention is added to a plate on which DNA of a target transcription regulatory region and a partial fragment thereof are immobilized, and direct binding between the two is determined by SPR (Surface Plasmon Resonance) method. Can be used for detection. In this case, the DNA of the target transcription regulatory region recognized by the transcription-related protein or a partial fragment thereof is immobilized on the sensor chip. The annealed DNA and its partial fragments are immobilized. As described above, the binding of the protein of the present invention or the DNA of the target transcriptional regulatory region and its partial fragments can be detected using fluorescent labels.
[0090]
The test sample for analysis containing the protein of the present invention can be prepared by the method of transcribing / translating the DNA of the present invention by an appropriate method as described above. Specifically, for example, protein expression can be induced by subjecting the DNA of the present invention to a cell-free transcription / translation system to obtain the protein of the present invention.
[0091]
In addition, a protein associated with transcription is analyzed for its effect on a transcription factor that regulates the transcription of various genes involved in a disease (hereinafter, referred to as “disease-related transcription factor”). It is also useful to analyze whether or not they are directly or indirectly involved. Known disease-related transcription factors include, for example, PPAR, p53, NFκB, AP-1, HIF-1, CREB, and the like. The effects on these known factors can be analyzed using the same method as described above. Just fine.
[0092]
For example, when analysis is performed using the SPR method, DNA of the target transcription regulatory region of the disease-related transcription factor or a partial fragment thereof is particularly preferably used. The test sample containing the protein of the present invention is appropriately added to the sensor chip on which such DNA or partial fragment is immobilized, and the interaction between the DNA on the sensor chip surface and the protein contained in the test sample is performed. Is analyzed. When it has an affinity with the immobilized DNA, the SPR response value is higher than that of the control, and it can be estimated that the transcription-related factor has an affinity with the immobilized DNA. By such an analysis, it is possible to analyze what function the protein of the present invention has in a specific disease.
[0093]
Here, the test sample for analysis containing the protein of the present invention can be prepared by the method of transcription / translation of the DNA of the present invention by an appropriate method as described above. Specifically, for example, protein expression can be induced by subjecting the DNA of the present invention to a cell-free transcription / translation system to obtain the protein of the present invention.
[0094]
The disease-related transcription factor can be obtained from a cell extract or a cell nucleus extract containing the disease-related transcription factor. In addition, as described above, it can be prepared by a method of transcription / translation of a DNA encoding a disease-related transcription factor by an appropriate method. Specifically, for example, a cDNA encoding a disease-related transcription factor is used, and its full length or a part thereof is inserted into an appropriate expression vector, and this is inserted into microorganisms such as Escherichia coli, insect cells, yeast, animal cells or animals. The disease-related transcription factor, which is a recombinant protein, can be obtained from the culture supernatant of these transfected cells into which the disease-related transcription factor has been expressed, or from cells, tissues, or body fluids.
[0095]
Note that confirmation of the activity of the protein of the present invention is not limited to the method described above. In addition, these functional assay systems can also be used for screening of a function activator or a function inhibitor of the protein of the present invention described later, and a screening of a protein expression regulator of the present invention.
[0096]
In general, the method for analyzing the function of the protein of the present invention includes, for example, (i) a method for comparatively analyzing the expression state of each tissue, disease, or developmental stage, and (ii) an interaction with another protein or DNA. A method of analyzing, (iii) a method of introducing into a suitable cell or individual, and analyzing a phenotypic change, and (iv) a method of analyzing the phenotypic change by inhibiting the expression of the protein in a suitable cell or individual. And the like. According to such a method, the activity specific to the target protein can be analyzed from multiple aspects.
[0097]
In the method (i), the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level. When the expression level is analyzed at the mRNA level, for example, an in situ hybridization method (In situ hybridization: Application to Developmental Biology & Medicine., Ed. by Harry, N. ed. 1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. In the case of analysis at the protein level, a tissue staining method using an antibody that specifically binds to the protein of the present invention described later, an ELISA method, a Western blot method, and the like can be mentioned. Here, when the protein to be analyzed is a splicing variant in which a known variant is present, a probe that is present only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA encoding the known variant should be used. Is preferred. In the case of the quantitative PCR method, a method of selecting and performing a primer capable of producing an amplified fragment having a different length between the target variant and the known variant (Wong, Y., Neuroscience Let., 320: 141-145 (2002)), etc. Is mentioned. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.
[0098]
In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As a method for analyzing the interaction, a conventional method known per se can be used, and specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal display And the like. In the method, when the protein to be analyzed is a splicing variant in which a known variant is present, the known variant is similarly analyzed for interacting substances, and a substance that specifically interacts with the target protein is identified. Is preferred.
[0099]
In the method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells and the like are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the introduced cells can be observed with a microscope, such as cell viability, cell growth rate, cell differentiation, neurite outgrowth when cells are neurons, localization and migration of intracellular proteins, etc. And those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins in cells. In the case of a splicing variant in which a known variant exists, these phenotypes can be similarly introduced into cells, and a phenotype related to the variant to be analyzed can be identified by comparative analysis. In addition, since it is known that the protein of the present invention has a DNA binding activity, it is also preferable to analyze by paying attention to the phenotype and the like found in diseases associated with the DNA binding protein.
[0100]
The method (iv) can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method. In this method, if a known variant is present in the target protein to be analyzed, a similar analysis is performed for the known variant and other variants, and a function specific to the target protein is identified by comparative analysis. Can be.
[0101]
(6) Screening for a molecule that regulates the activity of the protein of the present invention
By screening for a substance that specifically binds to the protein of the present invention and has an action of inhibiting, antagonizing, or enhancing the function (activity) of the protein of the present invention, a function modulator of the protein of the present invention (hereinafter, referred to as (Sometimes referred to as "modulators").
[0102]
This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein. For example, a method of first contacting a protein of the present invention with a test substance and selecting the test substance based on the binding property to the protein as an index, and then selecting a test substance using the change in the activity of the protein of the present invention as an index. Can be used.
[0103]
The test substance may be any substance as long as it interacts with the protein of the present invention and may affect the activity of the protein. , Proteins, non-peptidic compounds, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method Phage display method, ribosomal display method, or the competition analysis method with the antibody described in the above (4). A substance found to bind to the protein of the present invention by such a method is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator is identified.
[0104]
As a specific analysis method, for example, when analyzing a substance that regulates DNA binding activity, the method described in the above (5) can be used. If the binding to double-stranded DNA is increased as compared to the absence of the substance, the substance may function as a DNA-binding activator, and may be reduced or inhibited. Can identify that the substance may function as a DNA binding inhibitor. Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homologous protein or orthologous protein for the DNA or recombinant protein of the present invention to be used. Furthermore, the substances screened by the above method may be selected as drug candidates by screening in vivo.
[0105]
The DNA-binding activity of the protein of the present invention includes, for example, a function of regulating gene expression by changing the DNA structure, and includes a gene expression regulating protein, a transcription factor, and the like. Transcription factors are signaling pathways on pathways related to cancer, signaling pathways on myocardial development, signaling pathways controlling sperm differentiation and motility, pathways controlling germ cell differentiation Signaling function, signaling function on pathways that regulate cell differentiation, function to generate glycerol triphosphate, signaling function on pathways that control the development, differentiation, proliferation, and survival of neurons, onset of Alzheimer's disease In addition to signal transduction functions on pathways that control DNA, various signaling pathways that control the development, differentiation, growth, proliferation, survival, regeneration, and cell functions of various cells It is a factor that binds to and regulates gene expression. Therefore, it can be used as a target for screening for therapeutic agents for various diseases related to these signal transductions. Compounds that can be identified by the present screening method include anticancer agents, therapeutic agents for diabetes, anti-inflammatory agents, therapeutic agents for neurodegenerative diseases, therapeutic agents for heart diseases, therapeutic agents for infertility, agents for regenerating tissue, therapeutic agents for Alzheimer's disease, therapeutic agents for obesity, It can be used as a therapeutic agent for diabetes, a therapeutic agent for cardiovascular diseases, a therapeutic agent for metabolic disorders, a therapeutic agent for anorexia, bulimia, and the like.
[0106]
Such modulators can be used alone for the clinical application, but can also be used as pharmaceutical compositions by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0107]
(7) Screening of the DNA expression regulator of the present invention
Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the same in the presence of a test substance. Specifically, for example, cells expressing the protein of the present invention described in (2) above are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. And the like, or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blotting, or the like.
[0108]
As the test substance, those described in the above (6) can be used. According to this analysis, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance increases as compared with the amount of the test substance, the substance functions as a substance for promoting the expression of the DNA of the present invention. If it is possible and, on the contrary, decreases, it can be determined that the substance can be used as a substance inhibiting the expression of the DNA of the present invention.
[0109]
The above-mentioned active ingredient can be used alone for clinical application, but can also be used as a pharmaceutical composition by blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0110]
(8) The DNA-introduced animal of the present invention
The transfected DNA containing the DNA of the present invention described in the above (1) is constructed, introduced into a fertilized egg of a mammal other than a human, and this is transplanted into a female individual uterus to generate the DNA. A non-human mammal into which DNA has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is extracted from an oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg. And so on. Thereafter, after culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth. Whether or not the target DNA has been introduced into the newborn can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
[0111]
The thus-obtained DNA-introduced animal of the present invention obtains its offspring by crossing this individual and subculturing it in a normal breeding environment while confirming that the introduced DNA is stably retained. be able to. In addition, the offspring can be obtained by repeating in vitro fertilization, and the strain can be maintained.
The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.
[0112]
(9) Other uses of the protein of the present invention and DNA containing a nucleotide sequence encoding the same
The protein of the present invention can be used as a carrier having it bound on a substrate. In addition, the nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 13 or 35 and a partial fragment thereof are described in any of SEQ ID NOs: 14 to 26 or 36. And its partial fragments can be used as a carrier on which they are bound on a substrate. These may be hereinafter referred to as “protein chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips or DNA chips or arrays may contain other proteins and DNAs in addition to the proteins and DNAs of the present invention.
[0113]
Here, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a substrate for binding proteins and DNA. When using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of the protein or DNA to the base can be easily carried out by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
[0114]
In addition, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. Here, the nucleotide sequence of the DNA includes the nucleotide sequence of the corresponding RNA. That is, a database of amino acid sequences and nucleotide sequences can be constructed by storing the obtained amino acid sequences and nucleotide sequences in an appropriate recording medium in a computer-readable predetermined format. This database may contain the base sequences of other types of proteins and DNAs encoding them. Further, in the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Suitable recording media include, for example, magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RAM; and semiconductor memories. And the like.
[0115]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1  Preparation of cDNA library
(1) Preparation of mRNA
mRNA-prepared mouse (C57BL / 6) 0.5 to 1 g of each organ or tissue is homogenized with 10 ml of a suspension, and 1 ml of 2M sodium acetate at pH 4.0 and the same amount of phenol / chloroform (5: 1 by volume). The mixture was added and extracted. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes. The sample was washed with 70% ethanol, dissolved in 8 ml of water, and added with 2 ml of 5 M NaCl, 1% of CTAB (cetyltrimethy-lammonium bromide), 4 ml of urea, and 16 ml of an aqueous solution of pH 7.0 containing 50 mM Tris to remove RNA. The precipitate was removed to remove the polysaccharide (CTAB precipitation).
[0116]
Subsequently, the RNA was centrifuged at 4,000 rpm for 15 minutes at room temperature to dissolve the RNA in 4 ml of 7M guanidine-C1. After adding twice the volume of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to collect RNA, which was dissolved again in water. RNA purity was measured by reading the OD ratios 260/280 (> 1.8) and 230/260 (<0.45).
[0117]
(2) Preparation of first strand cDNA
Using 15 μg of the mRNA prepared in (1) above, 5-methyl-dCTP, dATP, dTTP, and dGTP were converted to 0.54 mM and 0.6 M trehalose in a reaction volume of 165 μl using reverse transcriptase 3,000 units. The reverse transcription reaction was performed under the following conditions: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2, 10 mM DTT, 52 ng / μl BSA, and RNase inhibitor 5 units. Oligonucleotide (SEQ ID NO: 27) containing a recognition sequence for restriction enzyme XhoI (wherein V represents A, G, or C, and N represents A, G, C, or T) 12.6 μl is used as a primer. Was.
[0118]
At the start of the reaction, 1/4 of the reaction solution is collected and 1.5 μl of [α-³²By adding P] -dGTP (3000 Ci / mmol, 10 μCi / μl; manufactured by Amersham), the synthesis efficiency of the first strand cDNA was measured. 0.5 μl of the RI-labeled reaction solution was spotted on DE-81 paper, and the RI activity before and after washing three times with 0.5 M sodium phosphate buffer (pH 7.0) was measured and calculated. Thereafter, the RI-labeled reaction solution and the non-labeled reaction solution were mixed, 8 μl of 0.5 M EDTA, 2 μl of 10% SDS, and 20 μg of proteinase K were added, and the mixture was heated at 45 ° C. for 15 minutes. After extraction with phenol / chloroform and ethanol precipitation, the precipitate was dissolved in 47 μl of RNase-free water (hereinafter referred to as RNase-free water).
[0119]
(3) 5 'cap structure and addition of biotin to 3' end
Biotinylated RNA Diol In order to bind biotin to the diol site (present at both the 5 'end of the Cap structure and the ribose at the 3' end of the poly A chain), a two-step reaction was performed. They are the oxidation of a diol group followed by the coupling reaction of biotin hydrazide with an oxidized RNA. First, 15 μg of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was placed in a 50 μl reaction mixture using a 6.6 mM sodium acetate buffer (pH 4.5) and sodium periodate as an oxidizing agent. Processed. This oxidation reaction was performed on ice under light-shielding conditions for 45 minutes.
[0120]
Subsequently, 11 μl of 5 M sodium chloride, 0.5 μl of 10% SDS, and the same amount of isopropanol were added, left on ice for 60 minutes, and centrifuged at 4 ° C. for 15 minutes at 15,000 rpm to obtain a precipitate. The precipitate was washed with 70% ethanol and redissolved in 50 μl of RNase-free water. 5 μl of 1 M sodium acetate (pH 6.1), 5 μl of 10% SDS, and 150 μl of 10 mM biotin hydrazide (manufactured by Sigma) were added to the sample, and reacted overnight at room temperature (22 to 26 ° C.). Finally, 5 μl of 5 M NaCl, 75 μl of 1 M sodium acetate (pH 6.1) and 2.5 volumes of ethanol were added, and the mixture was cooled on ice for 1 hour, centrifuged at 4 ° C. for 15 minutes, and biotinylated. After the reaction, the reaction solution was centrifuged for 15 minutes to precipitate the RNA-DNA complex again. The precipitate was washed once with 70% ethanol and once with 80% ethanol, and dissolved in 70 μl of RNase-free water.
[0121]
(4) Selection of full-length cDNA by RNase I
By treating the biotinylated RNA-DNA complex obtained in the above (3) with RNase I that digests single-stranded RNA, mRNA whose mRNA was not completely elongated during the reverse transcription reaction, and mRNA of mRNA The biotin residue labeled at the 3 'end was removed. Specifically, 10 μl of 10 × RNase I buffer (100 mM Tris-HCl (pH 7.5), 50 mM EDTA, 2 M NaOAc) was added to 70 μl of the sample obtained in (3), and RNase I (RNase One).^TM200 units (Promega), and the single-stranded RNA was digested at 37 ° C. for 15 minutes.
[0122]
(5) Collection of full-length cDNA
To prevent non-specific adsorption of cDNA to magnetic beads coated with streptavidin, 100 μg of yeast tRNA (treated with DNase I) was added to 5 mg (500 μl) of magnetic beads glass (MPG) particles coated with CPT. , NJ)) and left on ice for 1 hour, followed by washing with a solution of 50 mM EDTA and 2 M NaCl.
[0123]
The beads were suspended in 500 μl of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) above was added. By stirring for 30 minutes at room temperature, the magnetic beads and the full-length cDNA were bound. The beads capturing the full-length cDNA were washed 4 times with a solution of 50 mM EDTA and 2 M NaCl, and once with 0.4% SDS, 50 μg / μl yeast tRNA, 10 mM NaCl, 0.2 mM EDTA, and 10 mM Tris-HCl (pH 7.5). Once with 20% glycerol, once with a 50 μg / μl aqueous solution of yeast tRNA, a buffer for RNase H (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2₂, 20 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol (DTT)), suspended in 100 μl of RNase H buffer, added with 3 units of RNase H, and heated at 37 ° C. for 30 minutes. Thereafter, 1 μl of 10% SDS and 2 μl of 0.5 M EDTA were added, and the mixture was exposed to 65 ° C. for 10 minutes, and the supernatant was collected.
[0124]
The thus recovered single-stranded full-length cDNA was extracted with phenol / chloroform, and the volume of the solution was reduced to 100 μl or less using a speed bag, and then subjected to G25 / G100 Sephadex chromatography. The fraction having RI activity was collected in a silicon-treated microtube, and 2 μg of glycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 μl of ultrapure water.
[0125]
(6) Addition of oligo dG to single-stranded cDNA
30 μl of the single-stranded cDNA recovered in the above (5) was mixed with 200 mM sodium cacodylate (pH 6.9), 1 mM MgCl 2 in a final volume of 50 μl of the reaction solution.₂, 1 mM CoCl₂Under the conditions of 1 mM 2-mercaptoethanol and 100 μM dGTP, oligo dG addition reaction was carried out at 37 ° C. for 30 minutes using 32 units of terminal deoxynucleotidyl transferase (TaKaRa). At the end of the reaction, EDTA was added to 50 mM and dissolved in 31 μl of ultrapure water through a series of extractions with phenol / chloroform and ethanol precipitation.
[0126]
(7) Second strand cDNA synthesis
The synthesis of the second-strand cDNA using the first-strand cDNA as a template was performed as follows. In a reaction system having a final volume of 60 μl, a second strand low buffer (200 mM Tris-HCl (pH 8.75), 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄3%, 1% Triton X-100, 1 mg / μl BSA, second strand high buffer (200 mM Tris-HCl (pH 9.2), 600 mM KCl, 20 mM MgCl₂) 3 μl, 0.25 mM each of dCTP, dATP, dTTP, and dGTP, 6 μl of β-NADH, 31 μl of oligo dG-added first-strand cDNA, 600 ng of second-strand primer-adapter (SEQ ID NO: 28), and Ex Taq DNA polymerase ( Second-strand cDNA was synthesized using 15 units of TaKaRa ExTaq (TaKaRa), 150 units of thermostable DNA ligase (Ampligase; Epicentre), and 3 units of heat-resistant RNase H (Hybridase; Epicentre).
[0127]
The reaction was stopped by adding 1 μl of 0.5 M EDTA, and further heated at 45 ° C. for 15 minutes in the presence of 1 μl of 10% SDS and 10 μg of proteinase K to finally dissolve the protein components. A double-stranded full-length cDNA purified by extraction with ethanol / chloroform and ethanol precipitation was obtained.
[0128]
(8) Preparation of library
The double-stranded full-length cDNA obtained by the above method was inserted into a λZAPIII vector and recovered as a library. The λZAPIII vector is obtained by modifying SEQ ID NO: 29, which is a partial sequence of the multiple cloning site of λZAPII (manufactured by STRATAGENE) vector, to SEQ ID NO: 30 and newly introducing two SfiI sites.
[0129]
Further, a λPS (RIKEN) vector was prepared, and cDNA was inserted. λPS (RIKEN) (named λ-FLC-1 (FLC means FULL-LENGTH cDNA)) is a λPS vector of MoBiTec (Germany) modified for cDNA. That is, BamHI and SalI convenient for cDNA insertion are respectively introduced into cloning sites existing on both sides of a 10 kbp stuffer, and a 6 kb DNA fragment is inserted into an XbaI site so that a cDNA of about 0.5 kb to about 13 kb can be cloned. (JP-A-2000-325080). Using this λ-FLC-1, for example, in the case of a lung cDNA library, the average chain length of the insert was 2.57 kb, and it was possible to actually clone an insert of 0.5 kb to 12 kb. In the case of the conventional method λZAP, the average chain length of the insert was 0.97 kb, indicating that the use of λ-FLC-1 enables the cloning of a large-sized cDNA more efficiently than λZAP.
[0130]
Example 2  Normalization / subtraction of full-length cDNA library
(1) Preparation of driver
The mRNA prepared in Example 1 (1) (hereinafter sometimes referred to as “(a) RNA driver”) and the RNA prepared by in vitro transcription reaction were used as drivers. The latter RNA is further divided into two types (hereinafter referred to as "(b) RNA driver" and "(c) RNA driver"). One is obtained by recovering cDNA from RNA-cDNA removed by normalization and cloning into a phage vector. After infection with Escherichia coli, 1000 to 2000 plaques per starting material are mixed to form one library (mini-library), which is converted into plasmid DNA by a conventional method (the phage is infected again with Escherichia coli together with helper phage to form a phagemid). , And another infection to obtain plasmid DNA).
[0131]
The obtained DNA was subjected to an in vitro transcription reaction (using T3 RNA polymerase or T7 RNA polymerase), treated with DNase I (RQ1-RNase free; manufactured by Promega) and Proteinase K, and then extracted with phenol / chloroform to obtain RNA (b). An RNA driver was obtained. At this time, as a starting material, a mini-library is prepared from each of nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testes, small intestine, stomach), and the nine types of mini-libraries are mixed. To obtain RNA. As another RNA, a library (about 20,000 clones) already stored as a non-overlapping clone is cultured, and the obtained DNA is subjected to in vitro transcription reaction in the same manner as (b) RNA driver (c). ) RNA driver.
[0132]
These three kinds of RNAs were labeled with biotin using Label-IT Biotin Labeling Kit (manufactured by Mirus Corporation), added to tester cDNA at a ratio of 1: 1: 1, and reacted with Rot10 (42 ° C.). The second strand was synthesized with respect to the supernatant collected after the treatment with streptavidin beads (CPG).
[0133]
Example 3  Nucleotide sequencing of full-length cDNA clones
(1) clone rearray
One representative clone was selected from each cluster. Representative clones were selected with Q-bot (GENETIX LIMITED) and arrayed on a 384-well plate. At that time, E. coli was cultured in 50 μl of LB medium at 30 ° C. for 18 to 24 hours. At this time, when the cDNA library was introduced into the PS vector and transformed Escherichia coli DH10B, 100 mg / ml ampicillin and 50 mg / ml kanamycin were added, introduced into the Zap vector, and introduced into the SOLR system. If so, 100 mg / ml ampicillin and 25 mg / ml streptavidin were added.
[0134]
(2) Extraction of plasmid and InsSizing
Each of the clones cultured in the above (1) is further cultured in 1.3 ml of an HT solution containing 100 mg / ml of ampicillin, and the cells are collected by centrifugation. Then, QIAprep 96 Turbo (manufactured by QIAGEN) is used. To recover and purify the plasmid DNA. In order to examine the chain length of the cDNA inserted in the obtained plasmid, 1/30 of the plasmid DNA obtained above was digested with the restriction enzyme PvuII and subjected to 1% agarose gel electrophoresis.
[0135]
(3) Sequencing
Three types of sequencers were used to analyze the full-length nucleotide sequence of the full-length cDNA inserted into the thus obtained plasmid. In addition, plasmids were divided into two categories: those having insertion sequences shorter than 2.5 kb and those having longer insertion sequences. Among these, the nucleotide sequence of the clone having an insertion sequence shorter than 2.5 kb was analyzed from both ends. At this time, the plasmid was prepared using the primers described in SEQ ID NOS: 31 (sense strand) and 32 (antisense strand) when the vector was PS, and SEQ ID NO: 33 (sense strand) when the vector was Zap. , And 34 (antisense strand), and reacted with a Thermosequenase Primer Cycle Sequencing Kit (manufactured by Amersham Pharmacia Biotech), and analyzed using a Licor DNA4200 (long read sequencer).
[0136]
Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method. At this time, ABI Prism 377 and / or ABI Prism 3700 (manufactured by Applied Biosystems Inc.), BigDye terminator kit and Cycle Sequencing FS Ready Reaction Kit (Applied Systems, Inc.) were used.
[0137]
In addition, the sequence of a clone in which the inserted cDNA was longer than 2.5 kb was determined by the shotgun method. At that time, Shimadzu RISA 384 and DYEnamic ET terminator cycle sequencing kit (manufactured by Amersham Pharmacia Biotech) were used. To generate a shotgun library, 48 DNA fragments grown by PCR from 48 independent representative clones were used. The ends of the amplified DNA fragment were blunt-ended with T4 DNA polymerase.
This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector. A plasmid was prepared from this E. coli in the same manner as in the above (2).
[0138]
About those representative clones, the base sequence was determined by base sequence analysis from both ends, and the base sequences were ligated on a computer, and then subjected to sharing using Double Stroke Sharing Device (manufactured by Fire Inc.). Nucleotide sequence determination by the shotgun method was performed with duplication of 12 to 15 clones. The gaps whose sequence could not be determined by this nucleotide sequence determination were determined by primer walking in the same manner as described above.
[0139]
Example 4  Analysis of nucleotide sequence
(1) dnaform 34837 (SEQ ID NOS: 1, 14)
As shown in SEQ ID NO: 1, dnaform 34837 was composed of 3717 bases, of which base numbers 134 to 1690 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 518 amino acid residues (SEQ ID NO: 14). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 1 using BLAST, and the result was that (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). trebl | AK020600 | AK020600_1, RIKEN full-length enriched library, clone: 9530049C15, SCAN domain containing protein, e-value: 5 × 10^-171And (ii) the database registration symbol trembl | U88080 | U88080_1, Zinc finger protein protein 192 (LD5-1) is a Kruppel gene family with a SCAN box domain, and e-value: 2x10:⁻⁹⁴, 42%. In addition, (iii) the database registration symbol gp | AF154846 | 8099348, and the Homo sapiens zinc finger protein (ZFP) is e-value: 4 × 10⁻⁹³, 41% match. From these results, it was inferred that the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 was Zinc finger protein.
In addition, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 by using HMMPFAM. As a result, a sequence exhibiting the characteristics of ZF-C2H2 (a nucleotide sequence entered as LIM in Pfam) was found seven times. Was done.
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 was a zinc finger type transcription factor.
[0140]
(2) dnaform 63166 (SEQ ID NOs: 2, 15)
As shown in SEQ ID NO: 2, dnform 63166 was composed of 3607 bases, of which base numbers 612 to 2126 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 504 amino acid residues (SEQ ID NO: 15). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 2 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database). gpnew | BC021456 | 18204788, RIKEN cDNA 2810411K16, e-value: 2 × 10^-76100% identity over 139 amino acids, and (ii) the database registration symbol trembl | AK013086 | AK013086_1, RIKEN full-length enriched library, clone: 2810411K16: SCAN domain containing protein: 6 × -e:^-7699% identity over 139 amino acids, and (iii) the database accession gp | AK027384 | 14042023, Homo sapiens cDNA FLJ14478 fis, clone MAMMA1001633, e-value: 1 × 10⁻⁶¹, With 86% identity over 140 amino acids.
Further, when a protein characteristic search was performed by using HMMPFAM, a SCAN sequence was found at base numbers 1880 to 2167, and it was presumed from this characteristic that the protein was a DNA-binding protein.
[0141]
(3) dnaform 33383 (SEQ ID NOs: 3, 16)
As shown in SEQ ID NO: 3, dnform 33383 consists of 3704 bases, of which base numbers 662 to 3259 constitute an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 865 amino acid residues (SEQ ID NO: 16). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 3 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database). gp | BC008827 | 14250716, Homo sapiens, clone MGC: 13310 IMAGE: 4110431, e-value: 5 × 10^-145, 919 amino acids with a 40% identity, and (ii) the database entry symbol trembl | AF192913 | AF192913_1, Zinc finger protein 180 (HHZ168), e-value: 2 × 10⁻⁶³And (iii) the database registration symbol gp | U41164 | 1127843, Rattus norvegicus Cys2 / His2 zinc finger protein (rKr1), e-value: 6 × 10⁻⁶³, 56% identity over 192 amino acids.
In addition, a protein characteristic search by HMMPFAM was performed. As a result, six places of zf-C2H2 were found at base numbers 2786 to 3275, and it was inferred from these characteristics that the protein was a DNA-binding protein.
[0142]
(4) dnaform 39530 (SEQ ID NOs: 4, 17)
As shown in SEQ ID NO: 4, dnaform 39530 was composed of 2397 bases, of which base numbers 661 to 1476 were an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 271 amino acid residues (SEQ ID NO: 17). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 4 using BLAST, and the result was that (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). gp | AB05217 | 11611589, Macaca fascicularis brain cDNA, clone: QccE-18103. But e-value: 2 × 10^-95And 79% identity over 220 amino acids, and (ii) the database registration symbol trembl | AF017433 | AF017433_1, Homo sapiens putative transcription factor CR53 has an e-value of 1 × 10⁻³², 56%. (Iii) Database registration symbol gp | BC007287 | 13938317, Homo sapiens, Similar to zinc finger protein 202, clone MGC: 15660 IMAGE: 3347511, e-value: 1 × 10⁻³², With 56% identity over 134 amino acids.
In addition, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 by using HMMPFAM. As a result, an SCAN sequence was found at nucleotide numbers 775 to 1062 (a sequence to be entered as SCAN in Pfam).
From these, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 4 was a transcription factor.
[0143]
(5) dnaform 38861 (SEQ ID NOS: 5, 18)
As shown in SEQ ID NO: 5, dnform 38861 was composed of 2621 bases, of which base numbers 213 to 1703 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 496 amino acid residues (SEQ ID NO: 18). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 5 using BLAST. The homology search was performed in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database) into (i) a database registration symbol. gp | BC010591 | 14714873, Mus musculus, zinc finger proliferation 1, e-value: 6 × 10⁻⁹⁷, 491 amino acids with 41% identity, and (ii) the database registration symbol trmbl | D10630 | MMZFP51_1, mouse Zinc finger protein 38 (Zfp-38) (CtFIN51) (Transscription factor RU49), e-value: 10^-96And 41% identity over 491 amino acids, and (iii) the database entry symbol trembl | AB007886 | AB007886_1, Hypothetical zinc finger protein KIAA0426, e-value: 2 × 10^-95Hit with 44% identity over 532 amino acids.
Further, when protein characteristics were searched by HMMPFAM, zf-C2H2 was found eight times at base numbers 929 to 1585, and it was inferred from these characteristics that it was a DNA-binding protein.
[0144]
(6) dnaform 60441 (SEQ ID NOs: 6, 19)
As shown in SEQ ID NO: 6, dnform 60441 was composed of 1830 bases, of which base numbers 122 to 1612 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 496 amino acid residues (SEQ ID NO: 19). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 6 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). gp | BC010591 | 14714873, Mus musculus, zinc finger proferation 1, clone MGC: 18498 IMAGE: 3981599, e-value: 6 × 10⁻⁹⁷, 491 amino acids with 41% identity, and (ii) the database registration symbol trmbl | D10630 | MMZFP51_1, mouse Zinc finger protein 38 (Zfp-38) (CtFIN51) (Transscription factor RU49), e-value: 10^-96And 41% identity over 491 amino acids, and (iii) the database entry symbol trembl | AB007886 | AB007886_1, Hypothetical zinc finger protein KIAA0426, e-value: 2 × 10^-95Hit with 44% identity over 532 amino acids.
Further, when protein characteristics were searched by HMMPFAM, zf-C2H2 was found eight times at base numbers 929 to 1585, and it was inferred from these characteristics that it was a DNA-binding protein.
[0145]
(7) dnaform 42515 (SEQ ID NOs: 7, 20)
As shown in SEQ ID NO: 7, dnform 42515 consists of 2311 bases, of which base numbers 156 to 560 constitute an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 134 amino acid residues (SEQ ID NO: 20). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 7 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database). AJ301670, STATc transcriptional repressor, e-value: 4 × 10^-28And 53% identity over 127 amino acids, and (ii) the database registration code P58463, forkhead-related transcription factor 2, e-value: 4 × 10^-23And (iii) the database registration symbol P18480 and the Transcription regulatory protein SNF5 were e-value: 2 × 10⁻²⁴, With 61% identity over 118 amino acids.
Further, the protein of the above (i) is related to the control of the initial development rate and the timing of terminal differentiation from literature information (Mol. Cell, 7 (4), 779-88 (2001)) in the database, and , Can function as a repressor that regulates the expression of the ecmA gene. The protein of (ii) can be obtained from the literature information in the database (J. Biol. Chem. 1998, 273 (36): 23335-43). And that the transcription factor expressed in the placenta binds to the cis factor of several lung-specific genes. Furthermore, the protein of (iii) is described in the literature information (Mol. Cell. Biol., 1990, 10 (11): Transformation of genes regulated by glucose and phosphate from 5616-25) Be involved in the control has been revealed, respectively.
These results suggest that the protein encoded by the nucleotide sequence of SEQ ID NO: 7 is a transcription factor.
[0146]
(8) dnaform 41143 (SEQ ID NOs: 8, 21)
As shown in SEQ ID NO: 8, dnform 41143 consists of 2646 bases, and among them, base numbers 149 to 1591 constitute an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 480 amino acid residues (SEQ ID NO: 21). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 8 using BLAST, and it was found that (i) a database registration symbol was obtained in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database). AL03455, zinc finger protein 151 (pHZ-67), e-value: 1 × 10^-25And (ii) the database registration symbol Q13105, Myc-interacting zinc finger protein, with e-value: 2 × 10^-25Hit with 48% identity over 121 amino acids. From these results, it was inferred that the protein consisting of the amino acid sequence shown in SEQ ID NO: 21 was a Zinc finger type DNA binding protein.
In addition, the above-mentioned protein (ii) was found to be Myc-interacting zinc finger protein from literature information (Curr Top Microbiol Immunol 1997; 224: 137-46) in the database.
When a protein characteristic search was performed on the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 8 by HMMPFAM, a sequence showing characteristics related to protein dimerization at amino acids 9 to 121 (an amino acid entered as BTB in Pfam) Sequence) was found. In addition, Zinc finger domain (an amino acid sequence entered as zf-C2H2 in Pfam) was also found at one place.
From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 8 was a Zinc finger type DNA binding protein.
[0147]
(9) dnaform 34196 (SEQ ID NOS: 9, 22)
As shown in SEQ ID NO: 9, dnaform 34196 was composed of 2796 bases, of which base numbers 597 to 1982 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 461 amino acid residues (SEQ ID NO: 22). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 9 using BLAST. As a result, the SPTR protein database (a combination of the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database) contained (i) a database registration symbol. Q9P255, Hypothetical zinc finger protein KIAA1473, e-value: 1 × 10⁻⁵⁹And with 37% identity over 295 amino acids, and (ii) database entry P52742, Zinc finger protein 135 with e-value: 2 × 10^-58With 39% identity over 277 amino acids. From these results, it was inferred that the protein consisting of the amino acid sequence shown in SEQ ID NO: 22 was a Zinc finger type DNA binding protein.
The protein (ii) is known to be a zinc finger Kruppel family from literature information (Genomics 1995 May 20; 27 (2): 259-64) in a database, and is considered to be a developmental disorder. I found it involved.
In addition, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 9 by using HMMPFAM. As a result, Ninc finger domain (sequence entered as zf-C2H2 in Pfam) was found at 9 positions in amino acids 43 to 299. Found in.
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 9 was a Zinc finger type DNA binding protein.
[0148]
(10) dnaform 37479 (SEQ ID NOs: 10, 23)
As shown in SEQ ID NO: 10, dnform 37479 was composed of 2717 bases, of which base numbers 230 to 907 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 225 amino acid residues (SEQ ID NO: 23). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 10 using BLAST, and the result was that (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). Q9NQX1, PR-domain zinc finger protein 5 is e-value: 3 × 10^-13And (ii) the database registration symbol X82018, zinc finger protein with interaction domain, e-value: 2 × 10^-12, With 43% identity over 81 amino acids. From these results, it was inferred that the protein consisting of the amino acid sequence shown in SEQ ID NO: 23 was a Zinc finger type DNA binding protein.
The protein (ii) was found to be (zinc finger protein with interaction domain) from the literature information (Genes Dev 1994 Jul 15; 8 (14): 1664-77) in the database.
When the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 10 was searched for protein characteristics by HMMPFAM, Zinc finger domain at amino acids 12-34 and 40-63 (sequence to be entered as zf-C2H2 in Pfam) ) Were found in two places.
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 10 was a Zinc finger type DNA binding protein.
[0149]
(11) dnaform 59635 (SEQ ID NOS: 11, 24)
As shown in SEQ ID NO: 11, dnform 59635 was composed of 2709 bases, of which base numbers 566 to 1228 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 220 amino acid residues (SEQ ID NO: 24). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 11 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). Q9NQX1, PR-domain zinc finger protein 5 is e-value: 4 × 10^-13And (ii) the database registration symbol X82018, zinc finger protein with interaction domain, e-value: 3 × 10^-11, With 41% identity over 81 amino acids. From these results, it was inferred that the protein consisting of the amino acid sequence shown in SEQ ID NO: 24 was a Zinc finger type DNA binding protein.
The protein (ii) was found to be (zinc finger protein with interaction domain) from the literature information (Genes Dev 1994 Jul 15; 8 (14): 1664-77) in the database.
In addition, when a protein characteristic search was performed on the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 11 by HMMPFAM, amino acid numbers 12-34 and 40-63 showed Zinc finger domain (sequence to be entered as zf-C2H2 in Pfam). ) Was found.
From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 11 was a Zinc finger type DNA binding protein.
[0150]
(12) dnaform 33773 (SEQ ID NOs: 12, 25)
As shown in SEQ ID NO: 12, dnform 33773 consists of 3249 bases, of which base numbers 487 to 2127 constitute an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 546 amino acid residues (SEQ ID NO: 25). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 12 using BLAST. As a result, (i) a database registration symbol was found in the SPTR protein database (integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database). AF097416, Mus musculus zinc finger transcription factor Kaiso has e-value: 2 × 10⁻²⁷And (ii) the database registration number AF420316, Xenopus laevis BTB / POZ zinc finger transcription factor XKaiso, with an e-value of 2 × 10⁻²⁹Hit with 48% identity over 136 amino acids.
In addition, when a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 12 by using HMMPFAM, BTB (for BR-C, ttk and bab) or was found in the region of amino acid numbers 17 to 131 in SEQ ID NO: 25. A sequence (amino acid sequence entered as BTB in Pfam) showing the characteristics of the POZ (for Pox virus and Zinc finger) domain was found, and a sequence (Pfam) showing the characteristics of Msx-interacting-zinc finger in the region of amino acids 337-380. An amino acid sequence that is entered as zf-MIZ in the region of amino acid Nos. 340-508 was found at four positions showing the characteristics of Zinc finger, C2H2 type (Pfam). (An amino acid sequence entered as zf-C2H2).
From these results, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 is a Kaiso-like transcription factor having a zinc finger.
[0151]
(13) dnaform 51218 (SEQ ID NOs: 13, 26)
As shown in SEQ ID NO: 13, dnaform 51218 is composed of 2950 bases, of which a codon of an amino acid other than the stop codon continues up to 2950 (the last base of the sequence) in the frame following base number 167. The amino acid sequence encoded by the region from 167 to 2950 consists of 927 amino acid residues (SEQ ID NO: 26). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 13 using BLAST, and as a result, (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). AY044336, Xenopus laevis DNA-methylation dependent transcriptional repressor Kaiso-like protein (Kaiso) has e-value: 4 × 10⁻³²And with 48% identity over 137 amino acids, and (ii) the database registration number AF420316, Xenopus laevis BTB / POZ zinc finger transcription factor XKaiso, with an e-value of 4 × 10⁻³²Hit with a 48% match over 137 amino acids.
Further, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 by using HMMPFAM. As a result, BTB (BR-C, ttk and bab) or POZ was found in the region of amino acid numbers 17 to 131 in SEQ ID NO: 26. (Pox virus and Zinc finger) A sequence showing the characteristics of the domain (an amino acid sequence entered as BTB in Pfam) was found, and a sequence showing the characteristics of Msx-interacting-zinc finger in the region of amino acid numbers 337 to 380 (Pfam was zf) An amino acid sequence entered as MIZ) was found, and a sequence exhibiting the characteristics of Zinc finger, C2H2 type at five positions in the amino acid number 340-537 region (zf-C2H2 in Pfam) It has been found amino acid sequence), which is an entry in.
From these results, it was inferred that the nucleotide sequence shown in SEQ ID NO: 13 was a part of the sequence of the gene for a transcription factor of Kaiso-like having zinc finger.
[0152]
Example 5  Tissue expression analysis using DNA microarray
Tissue expression analysis is described in Miki, R .; , Et al. , Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).
(1) Preparation of DNA microarray
Nucleotide sequences of two types of mouse full-length cDNAs (dnaform 33383, dnaform 34196) and one type of mouse cDNA library FANTOM (HYPERLINK http: // hyperlink: http: //hyperlink.com) belonging to the same cluster as the mouse full-length cDNA to be analyzed (having a base sequence homologous to the cDNA). After amplifying the base sequence (FANTOM NO: 9530049C15) of cDNA derived from /fantom.gsc.riken.go.jp/http://fantom.gsc.riken.go.jp/ using M13 forward and reverse primers, The PCR product was precipitated with isopropanol and dissolved in 15 μl of 3 × SSC solution. These three DNA solutions were spotted on a glass slide coated with poly-L-lysine using a DNA arrayer of 16 chips (SMP3, TeleChem International, Sunnyvale, Calif.) To prepare a DNA microarray (for details of the method, see http ///Cmgm.stanford.edu/pbrown/mgide/index.html). Mouse β-actin and glyceraldehyde-3-phosphate dehydrogenase cDNA were used as a positive control, and Arabidopsis thaliana cDNA was used as a negative control.
[0153]
The detection sensitivity of this DNA microarray was 1 to 3 copies of mRNA per cell. The signal intensity of clones having approximately 80% identity with the target sequence was one-tenth that of clones with perfect sequence identity. The signal intensity of clones with less than 80% match with the target sequence was at the background level.
[0154]
(2) Preparation of probe
22 tissues of fetal, neonatal and adult C57BL / 6J mice (kidney, brain, spleen, lung, liver, testis, pancreas, stomach, small intestine, colon, placenta, heart, thymus, cerebellum, uterus, bone, muscle, spine 1 μg of mRNA extracted from side kidney-derived adipocytes, epididymal-derived adipocytes, visceral fat, 10-day-old neonatal cerebellum, 10-day-old neonatal skin) was subjected to a random prime reverse transcription reaction according to a standard method to obtain a fluorescent dye Cy3 (Amersham Pharmacia). I took it in. On the other hand, 1 μg of mRNA extracted from a whole body of a 17.5-day-old fetus was subjected to a random prime reverse transcription reaction, and the fluorescent dye Cy5 was taken in as a reference for expression analysis. The CyDye-labeled cDNA probe was purified using a CyScribe GFX Purification Kit (Amersham Pharmacia) and eluted from the column with 17 μl of sterile water. This was mixed with a blocking solution consisting of 3 μl of 10 μg / μl oligo (dA), 3 μl of yeast tRNA 20 μg / μl, 1 μl of 20 μg / μl mouse Cot1 DNA, 5.1 μl of 20 × SSC, and 0.9 μl of 10% SDS. Thus, a CyDye-labeled cDNA probe was prepared.
[0155]
(3) Hybridization of DNA microarray
A 30 μl solution obtained by mixing a cDNA probe (Cy3 label) derived from a tissue to be subjected to expression analysis and a reference cDNA probe (Cy5 label) derived from a 17.5-day-old fetus was heat-treated at 95 ° C. for 1 minute, and cooled at room temperature. The probe solution was added to the DNA microarray, covered with a cover slip, and hybridized at 65 ° C. overnight in Hybridasette (ArrayIt). Next, the DNA microarray was washed with 2 × SSC, 0.1% SDS, and subsequently rinsed with 1 × SSC for 2 minutes and 0.1 × SSC for 2 minutes. The microarray was scanned using a ScanArray 5000 confocal laser scanner, and the images were analyzed with IMAGENE (BioDiscovery).
[0156]
(4) Data analysis
The amount of mRNA (Cy3-labeled) in each tissue is expressed as a logarithm (log) of the ratio (Cy3 / Cy5) to the amount of fetal whole-body mRNA at 17.5 days of age (Cy5-labeled).₂). That is, if the mRNA expression level corresponding to each mouse full-length cDNA to be analyzed is larger in each tissue than in the reference tissue, it is a positive value; Indicated by Experiments were performed twice independently to increase the accuracy of the data and reproducible results were employed. The results are shown in Table 1 below.
[0157]
Generally, in expression analysis using a DNA array, an increase or decrease of about 2 times is regarded as an experimental error. From this, when the numerical value of the result shown in Table 1 is 1 or more, the amount of mRNA in a certain tissue is more than twice as large as the amount of mRNA of the whole body of a fetal 17.5-day-old control, which is significant. Was interpreted as increasing. Conversely, when the numerical value of the result is -1 or less, the amount of mRNA in a certain tissue is less than one-half that of the whole fetal mRNA at 17.5 days of age as a control, and is significantly reduced. I interpreted that. When comparing the mRNA expression levels between any tissues, if the difference between the values in each tissue is 1, the mRNA level is 2 times, and if it is 2, the mRNA level is 4 times. If the difference between the numerical values is -1, the amount of mRNA is 1/2 times, and if it is -2, the amount of mRNA is 1/4 times.
[0158]
Mouse cDNA clones belonging to the same cluster as the DNA spotted on the microarray and having a region having a nucleotide sequence identity of 80% or more over at least 200 bases are also described in Table 1 as cDNAs to be analyzed. And the numerical value of the measurement result of the DNA spotted on the microarray is described instead.
[0159]
[Table 1]

[0160]
Expression analysis of dnaform 34837 was found to increase only in the pancreas by expression analysis using FANTOM No: 9530049C15 as a probe. Expression analysis of dnaform 33383 using itself as a probe revealed that expression was reduced in a wide range of tissues. Expression analysis of dnaform 39530 was found to be reduced in a wide range of tissues by expression analysis using dnaform 33383 as a probe. Expression analysis of dnaform 34916 using itself as a probe revealed that expression was reduced only in testis.
[0161]
Example 6  Tissue expression analysis using PCR method
In order to examine the change in tissue expression of mRNA encoding the protein of the present invention in normal mice and diseased mice, a PCR method is used according to a standard method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)). The tissue expression analysis used was performed.
[0162]
(1) cDNA synthesis
Total RNA was extracted from 19 tissues of the following mice (Kazuo Moriwaki, one other edition, Molecular Medicine, separate volume, Vol. 36, “Spontaneous Disease Model Animals”, Nakayama Shoten, 1999), and reverse transcription was performed using oligo dT as a primer. CDNA synthesis was performed using the enzyme.
(A) Tissue of normal mouse and tissue of diabetic model mouse
{Circle around (1)} Control mouse C57BL / KsJ − + m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lung, kidney, bone marrow, pancreas, fat cells, liver, eyes
(2) Diabetes model mouse C57BL / KsJ-db / db Jcl (female, 8 weeks old) pancreas, adipocyte, liver, eye
(B) Tissue of senescence accelerating mouse
(1) Normally aged mouse SAM R1 / TA Slc (male, 13 weeks old) hippocampus, frontal cortex
(2) Senescence-accelerated mouse SAM P8 / Ta Slc (male, 15 weeks old) hippocampus, frontal cortex
(C) Tissue of cancer metastasis model mouse
(1) Normal colon of control mouse Balb / c (female, 5 weeks old)
(2) Colon metastasis model mouse Balb / c (female, 6 weeks old) colon cancer (colon cancer cells Colon 26 are transplanted into the mouse abdominal cavity, and colon cancer is removed 2 weeks later)
[0163]
(2) Quantification by PCR method
The expression of the following six mRNAs encoding the protein of the present invention was attached to the product using a LightCycler quantitative PCR device (Roche Diagnostics) and a LightCycler-FastStart DNA master SYBR Green I reagent. Quantification was performed according to the protocol provided. The synthetic DNA sequences used for quantitative PCR are shown below.
[0164]
(A) dnaform 33773
5'-side primer: AGGATTGGCGAACTATCCAG (SEQ ID NO: 37)
3'-side primer: CCACGAGTGAACATTTGCAT (SEQ ID NO: 38)
(B) dnaform 38861
5'-side primer: ACTCAGAGAGCCACGCCAAAA (SEQ ID NO: 39)
3'-side primer: TGTTGGAACCGTTTCCTGAG (SEQ ID NO: 40)
(C) dnaform 41143
5'-side primer: GCTGCACAAACGGTCTCATA (SEQ ID NO: 41)
3'-side primer: ACCAAGAGGGGCAACAGGAG (SEQ ID NO: 42)
(D) dnaform 42515
5'-side primer: TGTGCTGTCATCTGAGACTTTGA (SEQ ID NO: 43)
3'-side primer: CCTTGTTACAACCAAGGGTAGA (SEQ ID NO: 44)
(E) dnaform 51218
5'-side primer: AGGATTGGCGAACTATCCAG (SEQ ID NO: 45)
3'-side primer: CTCCACGAGTGAACATTTGC (SEQ ID NO: 46)
(F) dnaform 60441
5'-side primer: TCAGGAAACGGTTCCAACAT (SEQ ID NO: 47)
3'-side primer: TCCTTGGAGGATTTCTTTCTCTG (SEQ ID NO: 48)
[0165]
The quantitative results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. That is, the expression amount (copy number / μl) of the target gene in each tissue was divided by the expression amount of GAPDH (copy number / μl) to obtain a constant (1 × 10⁶) (Note: 10 to the sixth power).
[0166]
To summarize the results, dnaform 33773 was strongly expressed in the whole body, but was particularly strongly expressed in lung, pancreas, fat and brain. Although dnaform 38861 was strongly expressed in the whole body, it was particularly strongly expressed in pancreas and lung. Dnaform 41143 was expressed systemically, but was relatively strongly expressed in pancreas, lung and fat. dnaform42515 was specifically expressed in the brain, especially in the frontal cortex. Although dnaform51218 is strongly expressed in the whole body, it was particularly strongly expressed in lung, pancreas, fat and brain. Although dnaform 60441 was expressed systemically, it was particularly strongly expressed in pancreas and lung. The cDNA of the clone and the protein encoded by the cDNA can be applied to the treatment and diagnosis of diabetes, cancer and the like. Further, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the expression of mRNA is fluctuated as described above or a tissue having a high mRNA expression level.
[0167]
[Table 2]

[0168]
Example 7  Protein-protein interaction analysis
Using the two-hybrid method in mammalian cells (Suzuki, H., et al., Genome Research, 11, 1758-1765 (2001)), two types of mouse full-length cDNA base sequence (dnaform33773, dnaform34837) proteins The protein-protein interaction of the protein encoded by the coding sequence was comprehensively analyzed.
(1) Rapid sample preparation using PCR method
In a two-hybrid experiment using mammalian cells, a CheckMate mammarian two-hybrid system (manufactured by Promega) was used. Samples for protein-protein interaction analysis were a plasmid vector pBIND having a Gal4 gene DNA binding region inserted downstream of a CMV promoter, a plasmid vector pACT having a VP16 gene transcription activation region inserted downstream of a CMV promoter, and 5 Was prepared using a plasmid vector pG5luc in which a reporter luciferase gene was inserted downstream of the Gal4 binding region and the TATA box. A fusion gene of the Gal4 gene and the protein coding sequence of the nucleotide sequence (dnaform 33773, dnaform 34837) of the two types of mouse full-length cDNAs, and the VP16 gene and the mouse cDNA library FANTOM (http://fantom.gsc.liken.go.jp/). ), A fusion gene with the protein coding sequence of the full-length cDNA of each clone was prepared basically by a combination of ligation using a common sequence and a two-step PCR method according to the protocol of Promega (Suzuki, H., et al.). et al., Genome Research, 11, 1758-1765 (2001)). The protein coding sequence of the mouse cDNA was PCR-amplified using a forward primer having a common sequence on the 5 ′ side and a gene-specific sequence on the 3 ′ side and an M13 universal primer, and then the above amplification product and pBIND or pACT were amplified. A PCR amplification product (with a common sequence added to the 3 ′ side) is mixed, and a second-stage PCR amplification is performed using nested primers to express a fusion protein of Gal4 and mouse protein (BIND sample) Alternatively, a vector (ACT sample) for expressing a fusion protein of VP16 and mouse protein was constructed.
[0169]
(2) Two-hybrid experiments on high-throughput mammalian cells
BIND and ACT samples prepared by the PCR method were used directly without further purification. 0.25 μl of each of the BIND sample and the ACT sample, 30 ng of pG5luc, and 9.5 μl of Opti-MEM medium (Lifetech) were dispensed into a 384-well plate. 10 μl of LF2000 transfection reagent (manufactured by Lifetech) diluted 32 times with Opti-MEM medium was added to the wells, mixed, incubated for 20 minutes, and then suspended in F12 medium at 1,300 cells / μl CHO-K1 Chinese. 20 μl of a hamster cell solution was added and well suspended. The assay sample is₂After culturing for 20 hours in an incubator, luciferase activity was measured using Steady-Glo Luciferase Assay System (Promega) to confirm the interaction.
[0170]
As a result, the proteins encoded by the protein coding sequences of the nucleotide sequences (dnaform 33773 and dnaform 34837) of the two types of mouse full-length cDNAs are the following specific proteins (protein coding sequences of cDNAs contained in specific clones of the mouse cDNA library FANTOM): Has a specific protein encoded by the protein.
[0171]
[Table 3]

[0172]
(3) Consideration
The protein of the present invention encoded by Dnaform 33773 was found to have an intermolecular interaction with SRP40. SRP40 is one of the SR proteins which are a group of proteins involved in the splicing reaction of the pre-mRNA, and is a protein necessary for the splicing pathway of protein kinase C beta II mRNA involving phosphatidylinositol 3-kinase. It is known that this SRP40 regulates the expression of protein kinase C beta II, and this action regulates insulin-dependent glucose metabolism (J. Biol. Chem., 276 (25): 22648-54). , 2001). Therefore, an abnormality in SRP40 may cause diabetes. The protein of the present invention encoded by Dnaform 33773 is expected to have a Zinfinger and to have a transcription control activity, and when these results are analyzed comprehensively, it is encoded by Dnaform 33773 that binds to SRP40 that can cause diabetes. Can be presumed to be a protein that can cause diabetes.
The protein of the present invention encoded by Dnaform 34837 was found to interact with Pregnane X receptor 2 (PXR2). PXR2 is one of the nuclear steroid receptors and one of the nuclear receptors that is involved in the steroid compound-mediated signal transduction system in a complex manner. In the liver, it is known to be present in the cytoplasm and form a heterodimer with RXR (Retinoid X receptor) upon exposure to a ligand to translocate to the nucleus and to be involved in specific gene expression. . Specifically, for example, PXR is known to induce a drug metabolism system such as a cytochrome P450 (CYP) enzyme in response to a drug, and it is known that the PXR functions as a foreign substance (drug) sensor in a living body. Is speculated. It is known that this PXR abnormality causes so-called lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, and ischemic heart disease, and causes various endocrine diseases. The protein of the present invention encoded by Dnaform 34837 is a Zinc-finger protein, which is predicted to be a protein having a transcription control activity, and which is present in the nucleus and binds to a specific DNA sequence to transmit information. Is expected to be involved. Comprehensive analysis of the above results suggests that Dnaform 34837 is a protein that may form a heterodimer under conditions coexisting with PXR2 and may be involved in transcriptional control.
[0173]
Example 8  Obtaining human ortholog DNA
(1) Prediction of DNA ortholog DNA of dnaform33383
BLAST search for the human genome draft sequence (NCBI Build 30; http://www.ncbi.nlm.nih.gov/About/Doc/hs#genomeintro.html) using the base sequence of dnaform33383 (SEQ ID NO: 3) as a query. As a result, a region from 24.62 Mb to 24.66 Mb of 6p12.1 on chromosome 16 was found as a region of high homology.
Gene region prediction was performed on the genomic sequence region using a gene prediction program Genscan (http://genes.mit.edu/GENSCAN.html), and homology search was performed on the obtained predicted transcript sequence. As a result, a sequence consisting of 3520 bases (SEQ ID NO: 49) was obtained. This nucleotide sequence contained an open reading frame (SEQ ID NO: 35) of 2904 nucleotides at nucleotides 319-3219. The mouse nucleotide sequence of SEQ ID NO: 3 was found to have 81% homology to the human nucleotide sequence of SEQ ID NO: 35 over a length of about 2580 base pairs.
[0174]
It was predicted that the nucleotide sequence of SEQ ID NO: 35 would be translated into the human amino acid sequence of SEQ ID NO: 36. There was 81% homology over the 864 amino acid sequence between the amino acid sequence of SEQ ID NO: 36 and the amino acid sequence predicted from the open reading frame of dnaform 33383 (SEQ ID NO: 16).
Further, when the amino acid sequence of SEQ ID NO: 36 was queried, a homology search by BLAST was performed on the mouse cDNA library FANTOM database (http://fantom.gsc.ricken.go.jp/), and the result was SEQ ID NO: 16. Has the highest homology.
[0175]
In addition, in the BLAST homology search with respect to the public database “emble” database and the patent database “Genseq” database, a human base sequence having a higher homology than the base sequence of SEQ ID NO: 35 could not be searched.
Therefore, SEQ ID NO: 35 was considered to be a novel human orthologous DNA for SEQ ID NO: 3.
(2) Analysis of the nucleotide sequence of the obtained human ortholog DNA
Further analysis was performed on the nucleotide sequence of the human ortholog DNA predicted in (1) above and the amino acid sequence encoded by the nucleotide sequence.
First, a homology search was performed on the amino acid sequence represented by SEQ ID NO: 36 using BLAST, and the NRDB protein database (a database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) In addition, there was no sequence showing homology to the amino acid sequence of the mouse described in SEQ ID NO: 16 in the database of patent sequences. From this, it was inferred that the protein having the amino acid sequence of SEQ ID NO: 36 was a human ortholog protein of a mouse protein having the amino acid sequence of SEQ ID NO: 16.
The human amino acid sequence described in SEQ ID NO: 36 was subjected to protein characteristic search using HMMPFAM. As a result, SCAN domain (amino acid sequence entered as SCAN in Pfam) was substituted for amino acids 39 to 134, and KRAB box was substituted for amino acids 229 to 269. The region (amino acid sequence entered as KRAB in Pfam) is described in amino acid numbers 775-797, 803-825, 831-853, 859-881, 887-909, and 915-937, which show the characteristics of Zinc Finger, C2H2 type domain. A sequence (an amino acid sequence entered as Pfam as zf-C2H2) was found.
SCAN domain is a conserved motif found at the N-terminus of some C2H2-type Zn finger proteins, and is known to have a role in regulating oligomer formation and controlling transcription. Kruppel-associated box (KRAB) is found in about one-third of C2H2-type Zn finger proteins, is present at the N-terminal part, and acts to suppress transcription upon binding to DNA, and acts together with the Zn finger domain in cells. It is known to be involved in differentiation and development.
From the above, it was presumed that the protein having the amino acid sequence of SEQ ID NO: 36 was a C2H2-type Zn finger protein having a Kruppel-associated box (KRAB) and was a transcription factor involved in the suppression of transcription.
[0176]
(3) Obtaining human ortholog DNA of dnaform33383
Since a novel human ortholog DNA was predicted in the above (1) and (2), the cDNA was cloned as follows.
First, in order to select a tissue serving as a genetic resource for obtaining the cDNA, a human tissue-derived cDNA (manufactured by Clontech) was used as a template by using forward primer 1 (SEQ ID NO: 50) and reverse primer 1 (SEQ ID NO: 51). When the PCR was performed, amplification of the target DNA of about 150 bp was confirmed in the uterus, prostate, pituitary gland, brain, fetal brain, hippocampus, and thalamus. Forward primer 1 corresponds to base numbers 1294-1313 of SEQ ID NO: 35, and reverse primer 1 corresponds to base numbers 1443-1424 of SEQ ID NO: 35.
[0177]
Next, in order to obtain the full length of the open reading frame, PCR using forward primer 2 (SEQ ID NO: 52) and reverse primer 2 (SEQ ID NO: 53) was performed using cDNAs derived from these tissues as templates. These primers are primers designed using a sequence located outside the open reading frame in the human base sequence of SEQ ID NO: 49, and forward primer 2 corresponds to base numbers 253-272 of SEQ ID NO: 49, Reverse primer 2 corresponds to base numbers 3256-3237 of SEQ ID NO: 49.
[0178]
As a result of the PCR, 2.9 kb of DNA was amplified in the uterus, prostate, pituitary gland, fetal brain, hippocampus, and thalamus, which was consistent with the expected value. Of these, direct sequencing was performed using PCR-amplified DNA fragments derived from the uterus, prostate, and fetal brain as templates, and it was found that all three had the same sequence and that there was no mistake due to PCR. Therefore, the PCR amplified DNA fragment derived from the uterus was ligated to the pCR4Blunting-TOPO vector (manufactured by Invitrogen), and the plasmids derived from the three independent colonies obtained were sequenced. Exactly the same sequence as base Nos. 253-1256 (including the base sequence corresponding to the full length of the human open reading frame described in SEQ ID No. 35) was inserted.
From the above, it was shown that the expected human transcript (protein) was actually expressed, and it was found that the cDNA could be obtained. The thus obtained human homolog DNA was designated as ZG1001.
[0179]
When the amino acid sequence derived from the human base sequence (SEQ ID NO: 36) and the amino acid sequence derived from the mouse base sequence (SEQ ID NO: 16) are compared, amino acid numbers 64-913 on the human amino acid sequence are compared with those on the mouse amino acid sequence. The degree of identity was high at amino acid numbers 7-856, the human amino acid sequence was 59 amino acids longer at the N-terminus, and 36 amino acids longer at the C-terminus.
Furthermore, when the human base sequence (SEQ ID NO: 35) and the mouse base sequence (SEQ ID NO: 3) were mapped to the human genome and the mouse genome, respectively, both were encoded in seven exons. Each of the N-terminal and C-terminal-encoding portions was one exon from a portion with high coincidence to a portion with low coincidence. That is, it was found that the difference between the N-terminus and the C-terminus of these sequences was not the proper use of exons but the difference between species.
[0180]
Example 9  Analysis of binding ability to target transcription regulatory region
(1) Preparation of the protein of the present invention using a cell-free protein synthesis system
For a plasmid containing DNA encoding the protein of the present invention, Proc. Natl. Acad. Sci. USA, 99, 14652-14657 (2002), and a PCR reaction was performed to prepare a DNA fragment for transcription. Using this DNA as a template, a transcription reaction was performed using SP6 RNA Polymerase (manufactured by Promega) to synthesize mRNA, and the mRNA obtained by ethanol precipitation was purified. Protein synthesis using this mRNA is described in JP-A-2002-204689 and Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002), using a cell-free protein synthesis system by an overlay method. The translation solution (25 μl) used in the overlay cell-free protein synthesis system includes Proc. Natl. Acad. Sci. USA, 97: 559-564 (2000), 6 μl of wheat germ extract and the above-mentioned mRNA (0.02 nmol) were added and used, and the composition was 24 mM Hepes / KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 10 μg creatine kinase, ribonuclease inhibitor (20 units), 2 mM DTT, 0.4 mM spermidine, 0.3 mM amino acid type, 0.3 mM spermidine L 2.7 mM magnesium acetate, 100 mM potassium acetate, 5 μg wheat germ-derived tRNA, 0.05% Nonidet P-40 and 0.0 Consisting of 5% NaN3. The translation buffer was 31.3 mM HEPES / KOH (pH 7.8), 2.67 mM Mg (OAc) 2, 93 mM KOAc, 1.2 mM ATP, 0.257 mM GTP, 16 mM creatine phosphate, 2.1 mM DTT, 0 mM .41 mM spermidine, 0.3 mM L-type amino acid (20 kinds), 1 μM E-64, 0.005% NaN 3, 0.05% NP-40. In the cell-free protein synthesis by the overlay method, first, 125 μl of the translation buffer was added to a 96-well plate, and the translation solution was slowly overlaid from the bottom into each of the holes containing the translation buffer. The reaction was carried out by keeping the temperature in an incubator at 16 ° C for 16 hours.
[0181]
(2) SPR measurement method using sensor chip with immobilized target sequence
According to the description in BIAapplications handbook, chapter 4.4, the following 53 types of double-stranded DNAs biotinylated were separately immobilized on the sensor chip surface. The sensor chip used was an SA type (manufactured by Biacore), and the BIPRORE3000 (manufactured by Biacore) was used for SPR measurement and analysis.
[0182]
A known transcription factor, a designed base sequence of DNA or DNA fragment of a target transcription regulatory region (hereinafter, this may be referred to as a “target sequence”), or a target transcription commonly recognized by a plurality of transcription factors The relationship between the base sequence of the regulatory region DNA or DNA fragment (hereinafter, this may be referred to as “consensus target sequence”) is as follows.
[1] v-jun, c-jun, junB, junD, dJRA, c-fos, fosB1, fosB2, Fra-1, LRF-1, v-maf, mafG, NF-E2 p45, aNF-E2, fNF- E2, Nrf short form, GCN4, yAP-1, CREB-2, ATF-3, CRE-BP1, CRE-BP3, ATF-a, CREB-341, CREB-327, CREM, dCREB2, dCREB2-b, dCREB2- Consensus target sequence of c, dCREB2-d, dCREB2-q, dCREB2-r, dCREB2-s: TGATGACGT (SEQ ID NO: 54)
[2] Consensus target sequence of C / EBPalpha, C / EBPbeta, p34C / EBPbeta, CHOP-10: AAGTGGCGAAAGAGACA (SEQ ID NO: 55)
[3] VBP, Hlf, CPRF-2, EmBP-1b, EmBP-1b, GBF1, GBF2, GBF3, CPRF-1, TAF-1, HBP-1a, GBF9, GBF1, GBF12, CPRF-3, TGA1a, TGA1b , O2, STE4 consensus target sequence: AGAAGCACGTGGG (SEQ ID NO: 56)
[4] Consensus target sequence of OPI1, E2A, E47, ITF-2 / SEF2-1B, SEF-1A, MyoD, p42Tal-1: AACAGATGGGT (SEQ ID NO: 57)
[5] Target sequence of HEN-1: GGGGCGCAGCTGCGGCCC (SEQ ID NO: 58)
[6] AhR, Arnt consensus target sequence: GGGGATTGCCGTG (SEQ ID NO: 59)
[7] USF target sequence: GTCACGTGGT (SEQ ID NO: 60)
[8] Consensus target sequence of NF-1A1, NF-1A1.1, NF-1A6, NF-1B1, NF-1B1, NF-1B2, NF-1C2 / CTF-2, CTF-4, CTF-6: CTGTGGGGTTTTGCACGGGGCCA (SEQ ID NO: 61)
[9] RF-X1 target sequence: GGTAACATAGCAAC (SEQ ID NO: 62)
[10] Consensus target sequence of AP2alphaA / AP-2alpha1, AP2alpha2, AP2alpha3, AP2alpha4, AP2alphaB, AP2beta, AP2gamma: CGCCCCCCGGCG (SEQ ID NO: 63)
[11] GR target sequence: GGTACAAAATGTTCT (SEQ ID NO: 64)
[12] AR target sequence: AACATTATGTTTCT (SEQ ID NO: 65)
[13] Target sequence of ER: AAGGGGAAATGACCCCCC (SEQ ID NO: 66)
[14] Target sequence of RXR-alpha: GGTCATAGGGGT (SEQ ID NO: 67)
[15] PPARalpha target sequence: CTAGGGCAAAGGTCA (SEQ ID NO: 68)
[16] PPARgamma target sequence: GGTCAAAGGTCA (SEQ ID NO: 69)
[17] Target sequence of COUP-TF1, HNF-4alpha1, and HNF-4alpha2: TGAACTTTGA (SEQ ID NO: 70)
[18] Target sequence of CF1: GGGGTCACC (SEQ ID NO: 71)
[19] Consensus target sequence of GATA-1, GATA-2, GATA-3, GATA-4: CCAGATAAGG (SEQ ID NO: 72)
[20] AREA / NIT-2 target sequence: TATCTC (SEQ ID NO: 73)
[21] Sp1 target sequence: GGGGGGGGGGG (SEQ ID NO: 74)
[22] Target sequence of YY1: CGGCCATCTTGGGCT (SEQ ID NO: 75)
[23] Consensus target sequence of Egr-1, Egr-2, Egr-3: TGCGTGGGCG (SEQ ID NO: 76)
[24] Target sequence of Snail: CACCTGTTTTCA (SEQ ID NO: 77)
[25] Target sequence of CF2-II: GTATATATA (SEQ ID NO: 78)
[26] Evi-1 target sequence: AGATAAGATAA (SEQ ID NO: 79)
[27] Ikaros, consensus target sequence of MZF-1: TTGGGAGG (SEQ ID NO: 80)
[28] Target sequence of Tramtrack 69K: GGACCTGC (SEQ ID NO: 81)
[29] HOX9 target sequence: TGACAGTTTAACGA (SEQ ID NO: 82)
[30] Target sequence of CDP: CCAATAATCGAT (SEQ ID NO: 83)
[31] Target sequence of HNF-1A: GGTTAATGAATTAACCAC (SEQ ID NO: 84)
[32] Consensus target sequence of Nkx-2.2, Nkx-2.5, TTF-1: TTAAGTGGTT (SEQ ID NO: 85)
[33] Consensus target sequence of Oct-1A, Oct-1B, Oct-1C: ATGCAAAT (SEQ ID NO: 86)
[34] Consensus target sequence of Oct-2, Oct-2.1 / Oct-2B: TATTTGCAT (SEQ ID NO: 87)
[35] Pax-3, Pax-6 consensus target sequence: CGTCACGCTTTGA (SEQ ID NO: 88)
[36] Pax-1 target sequence: CCGTTCCCGCTCTAGATAT (SEQ ID NO: 89)
[37] Consensus target sequence of HSF1 (short), HSF2, dHSF, fungal HSF: AGAAAAGAAAAAGAAA (SEQ ID NO: 90)
[38] Consensus target sequence of c-Myb, A-Myb, v-Myb, P (long), P (short), C1 (long), C1 (short): AACGGGCCC (SEQ ID NO: 91)
[39] c-Ets-1 # p54, Ets-1 # deltaiV / VII, Ets-2, Elk-1, SAP-1, SAP-1b, Erg-1, p55erg, Fli-1b, E4TF1-60 / GABP -Alpha, consensus target sequence of E74A: GACAGGAAGTG (SEQ ID NO: 92)
[40] Target sequence of IRF-1, IRF-2: GAAAAGCGAAACC (SEQ ID NO: 93)
[41] Target sequence of p50: GGGGACTTTCC (SEQ ID NO: 94)
[42] Consensus target sequence of NF-ATc and NF-Atp: AGGAAAA (SEQ ID NO: 95)
[43] Consensus target sequence of p91, p84: GAATTCCGGGAAATGG (SEQ ID NO: 96)
[44] Consensus target sequence of STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6: TTTCCCGGGAAATG (SEQ ID NO: 97)
[45] Target sequence of p53: GGACATGCCCGGGGCATGTC (SEQ ID NO: 98)
[46] Target sequence of MEF-2A: CCTTAAAAAATA (SEQ ID NO: 99)
[47] SRF target sequence: CCATATATGGACAT (SEQ ID NO: 100)
[48] Target sequence of E2: AACCAAAAACGGTAA (SEQ ID NO: 101)
[49] Target sequence of TBP: TATAAAA (SEQ ID NO: 102)
[50] Target sequence of SRY, Sox-5, Sox-9: AAAAAACAATAGGG (SEQ ID NO: 103)
[51] Target sequence of mat-Mc: TCATTGTT (SEQ ID NO: 104)
[52] Consensus target sequence of CP1A, CP1B, CBF-C: CTGATTGGCTACC (SEQ ID NO: 105)
[53] Target sequence of AML1a: TTGTGGT (SEQ ID NO: 106)
[0183]
First, with respect to DNAs having the above-mentioned 53 types of target sequences (SEQ ID NOS: 54 to 106), a DNA having biotin added to its 5 ′ side and a DNA having a base sequence complementary thereto are individually annealed by a standard method, A total of 53 types of double-stranded DNA were prepared. On the other hand, each sensor chip used for analysis has a flow cell divided into four parts. Flow cell 1 was used as a control without any immobilization, and flow cells 2, 3 and 4 each immobilized three types of the double-stranded DNA prepared above. In order to keep the immobilization density of the DNA on the sensor chip constant, the value (D) obtained by dividing the increase in the SPR response value (ΔRU-DNA) by the DNA immobilization by the DNA molecular weight (MW) is constant in each flow cell. ΔRU-DNA was regulated. In the same manner, the remaining DNA was immobilized.
[0184]
After the preparation of the sensor chip as described above, the binding activity to the protein of the present invention encoded by the cloned cDNA was analyzed. Many reports have been made on the analysis of the binding activity between DNA and protein by the SPR method. In this example, the measurement was performed with reference to the measurement conditions of Molecular Microbiology, 36 (3), 557-569 (2000). First, a flow path in which the flow cells 1-2-3-4 are connected in series is set. The running buffer is allowed to flow therethrough at a constant flow rate (5 μL / min), and after the SPR measurement value is stabilized, the baseline value (SPR-baseline) of each flow cell is measured. Next, the protein solution is flowed at the same flow rate to form a specific bond between the protein molecule and the DNA chain. After the injection for a certain period of time, the SPR response value (SPR-bound) of each flow cell was measured.
[0185]
(4) Method of analyzing measurement results obtained by SPR method
The true binding amount (B) was obtained by subtracting the baseline value from the SPR response value ([SPR-bound]-[SPR-baseline]), and further, a standardized value (nB) was obtained.
From the measurement results, the proteins of the present invention encoded by cDNAs of 13 clones of dnaform 33383, 33733, 34196, 34837, 37479, 38861, 39530, 41143, 42515, 51218, 59635, 60441, and 63166 are the aforementioned [1] to [1] to [ 53] has binding activity to at least one of the DNAs having a total of 53 types of target sequences (SEQ ID NOS: 54 to 106).
[0186]
Example 10  Human tissue expression analysis
In Example 8, a human ortholog (ZG1001) was obtained for dnaform33383. Therefore, in order to examine the tissue expression variation of the mRNA encoding the human protein of the present invention in normal humans and disease patients, a standard method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)) is used. Tissue expression analysis using the PCR method was performed.
[0187]
(1) cDNA synthesis
The following mRNAs of 9 human tissues were purchased from Clontech, and cDNA was synthesized by performing a reverse transcription reaction using oligo dT as a primer.
Normal brain, normal hippocampus, normal thalamus, normal kidney, normal liver, normal pancreas, normal skeletal muscle, normal fat, normal spleen
The following cDNAs of the eight human tissues were purchased from Clontech.
Normal breast, breast cancer, normal colon, colon cancer, normal lung, lung cancer, normal stomach, gastric cancer
The following two human cDNAs were purchased from Biochain.
Normal frontal lobe, Alzheimer's disease frontal lobe
The following total RNA of one human tissue was purchased from Unitech, and mRNA was extracted before cDNA synthesis.
Normal peripheral blood leukocytes
[0188]
(2) Quantification by PCR method
Expression of mRNA encoding the human protein of the present invention (mRNA of human ortholog ZG1001 of dnaform 33383) was performed using a light cycler quantitative PCR device (manufactured by Roche Diagnostics) and a LightCycler-FastStart DNA master SYBR Green I reagent. And quantified according to the protocol attached to the product. The synthetic DNA sequences used for quantitative PCR are shown below.
5'-side primer: GCTCCGTCCACTGATAAACC (SEQ ID NO: 107)
3'-side primer: GCGGATAAAATTCGATGCCCTA (SEQ ID NO: 108)
The quantification results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. That is, the expression amount (copy number / μl) of the target gene in each tissue was divided by the expression amount of GAPDH (copy number / μl) to obtain a constant (1 × 10⁵) (Note: 10 to the fifth power). Table 4 shows the results.
[0189]
[Table 4]

[0190]
As is clear from Table 4, ZG1001 mRNA was expressed in pancreas, spleen, and brain, and increased in breast cancer.
From these results, the above cDNA and the protein encoded by the cDNA can be applied to treatment and diagnosis of cancer, Alzheimer's disease, and the like. Further, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the expression of mRNA is fluctuated as described above or a tissue having a high mRNA expression level.
[0191]
Example 11  Comprehensive analysis of proteins encoded by each full-length cDNA
(1) dnaform 34837 (SEQ ID NOS: 1, 14)
The protein encoded by the dnaform 34837 cDNA was identified as at least one of the target sequences as a result of a binding experiment using the SPR measurement device (manufactured by BIACORE) described in Example 9 for DNA having 53 target sequences. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. Expression analysis revealed that the gene was strongly expressed in adipocytes derived from pancreas and epididymis. Furthermore, protein-protein interaction analysis confirmed that the protein encoded by the cDNA interacted with PXR2, one of the nuclear steroid receptors.
From the analysis described in Example 4 and the results of these experiments, it is considered that this protein is a DNA-binding protein that exists in the nucleus and may interact with multimers or other proteins. Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatments and diagnoses.
[0192]
(2) dnaform 63166 (SEQ ID NOs: 2, 15)
The protein encoded by the cDNA of dnaform 63166 was identified as at least one of the target sequences as a result of a binding experiment with DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. Expression analysis revealed that the gene was strongly expressed in medulla oblongata and mammary gland.
From the analysis described in Example 4 and the results of these experiments, it is considered that this protein is a DNA-binding protein that exists in the nucleus and may interact with multimers or other proteins. Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. Can be applied to treatment and diagnosis
[0193]
(3) dnaform 33383 (SEQ ID NOs: 3, 16)
The protein encoded by the dnaform 33383 cDNA was identified as at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement apparatus (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. According to the results of the PFAM search described in Example 4, the present protein is a protein having six C2H2 type Zinc Finger structures at the C-terminal and having SCAN domain and KRAB domain involved in protein interaction at the N-terminal. . PreLoc localization prediction of protein intracellularly suggests nuclear localization.
From the analysis described in Example 4 and the results of these experiments, it is considered that this protein is a DNA-binding protein that may interact with multimers or other proteins expected to be present in the nucleus. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue having a high mRNA expression level. Applicable to diagnosis
[0194]
(4) dnaform 39530 (SEQ ID NOs: 4, 17)
The protein encoded by the cDNA of dnaform 39530 was identified as at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue having a high mRNA expression level. Applicable for diagnosis.
[0195]
(5) dnaform 38861 (SEQ ID NOS: 5, 18)
The protein encoded by the cDNA of dnaform 38861 was found to bind to at least one of the target sequences as a result of a binding experiment using the SPR measurement device (manufactured by BIACORE) described in Example 9 to DNA having 53 target sequences. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. Expression analysis revealed that the gene was strongly expressed in the whole body, especially in the pancreas and lung.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatments and diagnoses.
[0196]
(6) dnaform 60441 (SEQ ID NOs: 6, 19)
The protein encoded by the cDNA of dnaform 60441 contained at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. Expression analysis revealed that the gene was strongly expressed in the whole body, especially in the pancreas and lung.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatments and diagnoses.
[0197]
(7) dnaform 42515 (SEQ ID NOs: 7, 20)
The protein encoded by the dnaform42515 cDNA was found to bind to at least one of the target sequences as a result of a binding experiment for DNA having 53 target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. Expression analysis also revealed that it was specifically expressed in the brain, especially in the frontal cortex.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. And treatment and diagnosis of neurological diseases.
[0198]
(8) dnaform 41143 (SEQ ID NOs: 8, 21)
The protein encoded by the cDNA of dnaform 41143 was identified as at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence. From expression analysis, it was expressed in the whole body, but relatively strongly expressed in pancreas, lung and fat.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatments and diagnoses.
[0199]
(9) dnaform 34196 (SEQ ID NOS: 9, 22)
The protein encoded by the cDNA of dnaform34196 was found to bind to at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue having a high mRNA expression level. Applicable for diagnosis.
[0200]
(10) dnaform 37479 (SEQ ID NOs: 10, 23)
The protein encoded by the cDNA of dnaform 37479 was identified as at least one of the target sequences as a result of a binding experiment to DNA having 53 target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA-binding protein having selectivity for the bound target sequence.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue having a high mRNA expression level. Applicable to diagnosis
[0201]
(11) dnaform 59635 (SEQ ID NOS: 11, 24)
The protein encoded by the cDNA of dnaform 59635 was identified as at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA binding protein with selectivity for the bound target sequence.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which mRNA expression is fluctuated or a tissue having a high mRNA expression level. Applicable for diagnosis.
[0202]
(12) dnaform 33773 (SEQ ID NOs: 12, 25)
The protein encoded by the cDNA of dnaform 33773 was found to bind to at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA binding protein with selectivity for the bound target sequence. Expression analysis revealed that the protein was strongly expressed in the whole body, but was particularly strongly expressed in lung, pancreas, fat, and brain. Furthermore, the protein-protein interaction analysis confirmed that the protein encoded by the cDNA interacted with SRP40, which can cause diabetes.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the expression of mRNA is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatment and diagnosis of neurological diseases and the like.
[0203]
(13) dnaform 51218 (SEQ ID NOs: 13, 26)
The protein encoded by the cDNA of dnaform51218 was found to bind to at least one of the target sequences as a result of a binding experiment to DNA having 53 types of target sequences using the SPR measurement device (manufactured by BIACORE) described in Example 9 above. After binding, the protein was shown to be a DNA binding protein with selectivity for the bound target sequence. Expression analysis revealed that the protein was strongly expressed in the whole body, but was particularly strongly expressed in lung, pancreas, fat, and brain.
Based on the analysis described in Example 4 and the results of these experiments, this protein is expected to be present in the nucleus, and is considered to be a DNA-binding protein that may interact with multimers or other proteins. . Therefore, the cDNA and the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the expression of mRNA is fluctuated or a tissue expressing a large amount of mRNA, such as diabetes or cancer. It can be applied to treatment and diagnosis of neurological diseases and the like.
[0204]
【The invention's effect】
Since the protein of the present invention and the DNA encoding the same have a DNA binding activity and the like, a substance that regulates the activity can be screened using the protein or the DNA encoding the same, and diseases associated with the protein can be screened. It is useful for the development of medicines that can act on the like.
This application discloses a Japanese patent application filed on April 19, 2002 (Japanese Patent Application No. 2002-117840), a Japanese patent application filed on April 30, 2002 (Japanese Patent Application No. 2002-128418), and a Japanese Patent Application No. 2002-128418. (Japanese Patent Application No. 2002-352469), the contents of which are incorporated herein by reference. The contents of the documents cited in the present specification are also incorporated herein by reference.
[0205]
[Sequence list]

Claims (15)

The following protein (a) or (b):
(A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 14 to 26 or 36;
(B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of any of SEQ ID NOs: 14 to 26 or 36, and which has DNA binding activity. A DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein according to claim 1. A DNA according to any one of the following (a), (b) or (c):
(A) a DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 13 or 35;
(B) a protein having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence set forth in any one of SEQ ID NOs: 1 to 13 or 35, and having a DNA binding activity DNA encoding.
(C) having a base sequence capable of hybridizing under stringent conditions to a DNA having the base sequence of any one of SEQ ID NOs: 1 to 13 or 35 or a sequence complementary thereto, and having a DNA binding activity DNA encoding a protein. A recombinant vector comprising the DNA according to claim 2. A gene-introduced cell into which the DNA according to any one of claims 2 to 4 or the recombinant vector according to claim 5 has been introduced, or an individual comprising the cell. A protein according to claim 1, which is produced by the cell according to claim 6. A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any one of claims 2 to 4, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and An oligonucleotide selected from the group consisting of oligonucleotide derivatives of the sense or antisense oligonucleotide. An antibody or a partial fragment thereof that specifically binds to the protein according to claim 1. The antibody according to claim 9, wherein the antibody is a monoclonal antibody. The antibody according to claim 10, wherein the monoclonal antibody has an action of neutralizing the DNA binding activity of the protein according to claim 1 or 7. A method for screening for an activity-regulating substance of a protein, comprising bringing the protein according to claim 1 or 7 into contact with a test substance, and measuring a change in the activity of the protein due to the test substance. A method for screening a substance regulating the expression of a DNA, comprising bringing the test substance into contact with the gene-transfected cell according to claim 6, and detecting a change in the expression level of the DNA introduced into the cell. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1 and / or at least one or more nucleotide sequence selected from the nucleotide sequence of the DNA according to any one of claims 2 to 4. A computer-readable recording medium that stores information. A carrier to which the protein according to claim 1 and / or the DNA according to any one of claims 2 to 4 are bound.